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Mechanisms of soft palate closure in human embryos Mattson, Melanie 2013

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	 ?MECHANISMS	 ?OF	 ?SOFT	 ?PALATE	 ?CLOSURE	 ?IN	 ?HUMAN	 ?EMBRYOS	 ?	 ?	 ? by	 ?Melanie	 ?Mattson	 ?	 ?B.Sc.Hons,	 ?The	 ?University	 ?of	 ?Calgary,	 ?2005	 ?DDS,	 ?The	 ?University	 ?of	 ?Alberta,	 ?2009	 ?	 ?	 ?A	 ?THESIS	 ?SUBMITTED	 ?IN	 ?PARTIAL	 ?FULFILLMENT	 ?OF	 ?THE	 ?REQUIREMENTS	 ?FOR	 ?THE	 ?DEGREE	 ?OF	 ?	 ?MASTER	 ?OF	 ?SCIENCE	 ?in	 ?THE	 ?FACULTY	 ?OF	 ?GRADUATE	 ?AND	 ?POSTDOCTORAL	 ?STUDIES	 ?	 ?THE	 ?UNIVERSITY	 ?OF	 ?BRITISH	 ?COLUMBIA	 ?(Vancouver)	 ?	 ?	 ?	 ?August	 ?2013	 ?	 ?	 ??	 ?Melanie	 ?Mattson,	 ?2013	 ?	 ? 	 ? ii	 ?Abstract 	 ? 	 ?	 ?Objectives:	 ?The	 ?human	 ?secondary	 ?palate	 ?forms	 ?between	 ?6-??12	 ?weeks	 ?of	 ?gestation.	 ?There	 ?has	 ?been	 ?controversy	 ?as	 ?to	 ?whether	 ?palatal	 ?shelves	 ?in	 ?the	 ?soft	 ?palate	 ?join	 ?by	 ?fusion	 ?similar	 ?to	 ?the	 ?hard	 ?palate,	 ?or	 ?whether	 ?merging	 ?and	 ?proliferation	 ?of	 ?the	 ?mesenchyme	 ?at	 ?the	 ?posterior	 ?edge	 ?of	 ?the	 ?developing	 ?hard	 ?palate	 ?is	 ?the	 ?mechanism.	 ?The	 ?purpose	 ?of	 ?this	 ?study	 ?is	 ?to	 ?examine	 ?the	 ?mode	 ?of	 ?soft	 ?palate	 ?closure	 ?in	 ?a	 ?more	 ?representative	 ?sample	 ?than	 ?was	 ?used	 ?in	 ?the	 ?single	 ?previous	 ?study	 ?on	 ?which	 ?all	 ?textbooks	 ?are	 ?based.	 ?Methods:	 ?Serial	 ?sections	 ?of	 ?secondary	 ?palates	 ?from	 ?13	 ?human	 ?fetuses	 ?from	 ?54-??74-??days	 ?of	 ?development	 ?post	 ?conception	 ?were	 ?stained,	 ?photographed	 ?and	 ?imported	 ?into	 ?WinSurf	 ?3D	 ?software.	 ?Anatomical	 ?structures	 ?were	 ?traced	 ?including	 ?the	 ?palatal	 ?shelves,	 ?midline	 ?epithelial	 ?seam	 ?and	 ?palatine	 ?aponeurosis,	 ?the	 ?images	 ?aligned	 ?and	 ?then	 ?stacked	 ?to	 ?create	 ?a	 ?3D	 ?representation.	 ?Results:	 ?We	 ?analyzed	 ?the	 ?following	 ?numbers	 ?of	 ?specimens:	 ?54-??days-??2;	 ?57-??days-??4;	 ?59	 ?days	 ?-??2;	 ?64-??days-??1;	 ?67-??days-??1;	 ?70-??days?2;	 ?74-??days-??1.	 ?At	 ?54-??days,	 ?a	 ?midline	 ?seam	 ?is	 ?present	 ?close	 ?to	 ?the	 ?hard	 ?palate	 ?but	 ?more	 ?posteriorly	 ?the	 ?soft	 ?palate	 ?is	 ?open.	 ?Between	 ?57	 ?and	 ?59	 ?days	 ?a	 ?thick	 ?midline	 ?seam	 ?is	 ?observed	 ?throughout	 ?the	 ?soft	 ?palate.	 ?There	 ?is	 ?some	 ?variability	 ?between	 ?specimens	 ?such	 ?that	 ?the	 ?soft	 ?palate	 ?was	 ?closed	 ?early	 ?in	 ?one	 ?59	 ?day	 ?specimen	 ?and	 ?open	 ?in	 ?a	 ?67-??day	 ?specimen.	 ?One	 ?70-??day	 ?specimen	 ?had	 ?no	 ?seam	 ?whereas	 ?the	 ?other	 ?retained	 ?the	 ?seam.	 ?By	 ?74-??days	 ?the	 ?specimen	 ?had	 ?complete	 ?soft	 ?palate	 ?union	 ?with	 ?the	 ?presence	 ?of	 ?a	 ?continuous	 ?palatine	 ?aponeurosis.	 ?Overall,	 ?our	 ?sample	 ?included	 ?a	 ?total	 ?of	 ?7	 ?fetuses	 ?with	 ?a	 ?midline	 ?seam	 ?in	 ?the	 ?soft	 ?palate.	 ?	 ?Conclusions:	 ?The	 ?formation	 ?of	 ?a	 ?bilayered	 ?epithelial	 ?seam	 ?followed	 ?by	 ?breakdown	 ?of	 ?the	 ?seam	 ?and	 ?mesenchymal	 ?fusion	 ?is	 ?the	 ?primary	 ?mode	 ?of	 ?soft	 ?palate	 ?formation	 ?in	 ?humans.	 ?Epithelial	 ?seam	 ?removal	 ?is	 ?rapid	 ?and	 ?could	 ?explain	 ?why	 ?a	 ?seam	 ?was	 ?not	 ?observed	 ?in	 ?earlier	 ?studies.	 ?	 ?	 ?	 ?	 ?	 ?	 ? 	 ? iii	 ?	 ?Preface Human	 ?conceptuses	 ?were	 ?obtained	 ?from	 ?the	 ?University	 ?of	 ?Washington	 ?between	 ?1988	 ?and	 ?1991	 ?by	 ?Dr.	 ?V.	 ?M.	 ?Diewert.	 ?	 ?The	 ?work	 ?was	 ?carried	 ?out	 ?under	 ?UBC	 ?Human	 ?Ethics	 ?approval	 ?#	 ?H08-??02576.	 ?This	 ?protocol	 ?was	 ?approved	 ?Nov.	 ?18,	 ?2011	 ?and	 ?is	 ?renewed	 ?annually.	 ?	 ?Work	 ?was	 ?supported	 ?by	 ?Faculty	 ?of	 ?Dentistry	 ?research	 ?funds	 ?to	 ?JMR.	 ?	 ?	 ?	 ?	 ?	 ? 	 ? iv	 ?	 ?Table of Contents Abstract ............................................................................................................................... ii Preface ............................................................................................................................... iii List of Tables ..................................................................................................................... vi List of Figures ................................................................................................................... vii List of Abbreviations ....................................................................................................... viii Acknowledgements............................................................................................................ ix Chapter 1 - Introduction...................................................................................................... 1 Staging of human embryos ..........................................................................................................1 1.1. Development of the primary palate...................................................................................3 1.2. Secondary palate development..........................................................................................6 1.3.1 Embryonic period........................................................................................................6 1.3.2 Development of the hard palate in the fetal period.....................................................8 1.3.3 Development of the soft palate in the fetal period ......................................................8 1.3.4 Muscle development in the soft palate......................................................................12 1.3. Clefting of the palate.......................................................................................................13 1.4. Microforms of clefting ....................................................................................................14 1.5. Etiology of orofacial clefting ..........................................................................................15      1.6.     Aims................................................................................................................................18 Chapter 2 - Methods ......................................................................................................... 19 2.1. Preparation of specimens and processing into paraffin ..................................................19 2.2. Histochemical and immunohistochemical staining procedures ......................................20 2.3. 3D reconstruction of human fetuses at various stages of palatogenesis .........................22 Chapter 3: Results ............................................................................................................. 23 3.1. Overview of craniofacial development from 54 ? 74-days post conception ..................23 3.1.1 Tooth development...................................................................................................26 3.1.2 Cartilages..................................................................................................................26 3.1.3 Bone development ....................................................................................................27 3.1.4 Muscle development.................................................................................................28 3.2. Palate development according to stage of embryo..........................................................32 3.2.1 54-day specimens have incomplete fusion of the hard and soft palates...................32 3.2.2 57-day specimens have completed hard but not soft palate closure.........................36 3.2.3 59-70-day specimens have complete hard palates, variable soft palate development 40 3.2.4 74-day specimen has a fully developed soft palate and aponeurosis .......................43 3.2.5 3-D reconstructions confirm an epithelial seam is present in the soft palate ...........44 3.2.6 Palatine aponeurosis condenses near the tensor veli palatini between 59 and 67d ..47 Chapter 4 ?Discussion ...................................................................................................... 49 4.1. Revised model of soft palate development .....................................................................49 4.2. Comparative Anatomy - Do other mammals have a seam in the soft palate? ................52 4.3. Variability of palate development in human fetuses.......................................................53 4.4. Animal models give insights into the genes that are required for soft palate morphogenesis ...........................................................................................................................55  	 ? v	 ?4.5 Limitations of this study ..................................................................................................58 References......................................................................................................................... 61 	 ?	 ? 	 ? vi	 ?	 ?List of Tables 	 ?Table 3.1 Palate development in individual human embryos and fetuses ........................ 24	 ?Table 3.2 Comparison of crown-rump length of specimens from current study with Burdi and Faist 1967 study ......................................................................................................... 25	 ?Table 3.3 Fusion status of the hard and soft palate in University of Washington specimens.......................................................................................................................... 34	 ? 	 ? vii	 ?List of Figures Figure 1.1. Schematic of palate closure from Burdi and Faist, 1967. .............................. 10	 ?Figure 3.1. Overview of craniofacial development in the conceptuses used in this study........................................................................................................................................... 31	 ?Figure 3.2. Palate morphogenesis in a 54-day specimen. ................................................. 35	 ?Figure 3.3 Development of the palate in a 57-day fetus. .................................................. 38	 ?Figure 3.4  Palatal morphogenesis in a second 57-day specimen..................................... 39	 ?Figure  3.5. Palatal morphogenesis of a 64-day fetus. ...................................................... 42	 ?Figure  3.6. 3D reconstructions of the hard palate-soft palate junction............................ 46	 ?Figure 3.7. Palatal muscle development in 59 and 67-day fetuses. .................................. 48	 ?Figure  4.1. The mechanism of palate closure is fusion in both the hard and soft palate. 50	 ?	 ?	 ? 	 ? viii	 ?List of Abbreviations BMP:	 ?bone	 ?morphogenetic	 ?protein	 ?BSA:	 ?bovine	 ?serum	 ?albumin	 ?CL/P:	 ?cleft	 ?lip	 ?and	 ?palate	 ?CPO:	 ?cleft	 ?palate	 ?only	 ?DAB:	 ?detection	 ?and	 ?visualization	 ?of	 ?antibody	 ?binding	 ?ddH2O:	 ?double	 ?distilled	 ?water	 ?EDTA:	 ?Ethylenediaminetetraacetic acid	 ?EtOH:	 ?ethanol	 ?H&E:	 ?hematoxalin	 ?and	 ?eosin	 ?IgG:	 ?Immunoglobulin	 ?G	 ?lvp:	 ?levator	 ?veli	 ?palatini	 ?mee:	 ?medial	 ?edge	 ?epithelium	 ?mes:	 ?midline	 ?epithelial	 ?seam	 ?PBS:	 ?phosphate	 ?buffered	 ?saline	 ?PCNA:	 ?proliferating	 ?cell	 ?nuclear	 ?antigen	 ?PFA:	 ?paraformaldehyde	 ?SHH:	 ?Sonic	 ?Hedgehog	 ?tvp:	 ?tensor	 ?veli	 ?palatini	 ?TUNEL:	 ?terminal	 ?deoxynucleotidyl	 ?transferase	 ?nick-??end	 ?labeling	 ?VPI:	 ?velopharyngeal	 ?insufficiency	 ? 	 ? ix	 ?	 ?Acknowledgements 	 ?I	 ?would	 ?like	 ?to	 ?offer	 ?my	 ?sincerest	 ?thanks	 ?to	 ?Dr.	 ?Joy	 ?Richman	 ?for	 ?all	 ?her	 ?help	 ?and	 ?support	 ?over	 ?the	 ?past	 ?three	 ?years.	 ?Without	 ?her	 ?I	 ?would	 ?not	 ?have	 ?been	 ?able	 ?to	 ?complete	 ?this	 ?project.	 ?	 ?Many	 ?thanks	 ?to	 ?Dr.	 ?Virginia	 ?Diewert,	 ?Kathy	 ?Fu,	 ?and	 ?the	 ?members	 ?of	 ?the	 ?Richman	 ?lab	 ?who	 ?have	 ?helped	 ?me	 ?along	 ?the	 ?way.	 ?	 ?Special	 ?thanks	 ?are	 ?owed	 ?to	 ?my	 ?parents,	 ?whose	 ?have	 ?supported	 ?me	 ?throughout	 ?my	 ?years	 ?of	 ?education,	 ?both	 ?morally	 ?and	 ?financially.	 ? 	 ? 1	 ?CHAPTER 1 - INTRODUCTION Staging of human embryos 	 ? In	 ?order	 ?to	 ?compare	 ?my	 ?results	 ?to	 ?those	 ?of	 ?others,	 ?it	 ?is	 ?first	 ?important	 ?to	 ?review	 ?the	 ?challenges	 ?with	 ?staging	 ?human	 ?embryos.	 ?As	 ?we	 ?shall	 ?see,	 ?when	 ?microscopic	 ?anatomy	 ?is	 ?being	 ?analyzed	 ?there	 ?are	 ?considerable	 ?variations	 ?in	 ?development	 ?for	 ?a	 ?given	 ?chronological	 ?age.	 ?	 ?Therefore	 ?accurate	 ?staging	 ?is	 ?essential	 ?to	 ?compare	 ?my	 ?results	 ?to	 ?studies	 ?of	 ?others.	 ?There	 ?are	 ?many	 ?different	 ?ways	 ?by	 ?which	 ?a	 ?human	 ?embryo	 ?or	 ?fetus	 ?can	 ?be	 ?staged.	 ?It	 ?is	 ?of	 ?utmost	 ?importance	 ?that	 ?the	 ?age	 ?of	 ?conception	 ?be	 ?determined	 ?as	 ?accurately	 ?as	 ?possible	 ?for	 ?research	 ?purposes,	 ?as	 ?huge	 ?developmental	 ?changes	 ?can	 ?be	 ?seen	 ?in	 ?an	 ?embryo	 ?or	 ?fetus	 ?over	 ?the	 ?course	 ?of	 ?a	 ?few	 ?hours	 ?to	 ?a	 ?few	 ?days.	 ?	 ?A	 ?general	 ?estimate	 ?can	 ?be	 ?determined	 ?by	 ?the	 ?menstrual	 ?age,	 ?which	 ?is	 ?calculated	 ?based	 ?on	 ?the	 ?last	 ?reported	 ?menstruation.	 ?	 ?Menstrual	 ?age	 ?is	 ?a	 ?value	 ?commonly	 ?used	 ?by	 ?obstetricians.	 ?Assuming	 ?ovulation	 ?and	 ?fertilization	 ?occurred	 ?14	 ?days	 ?after	 ?the	 ?first	 ?day	 ?of	 ?the	 ?last	 ?menstruation,	 ?fertilization	 ?age	 ?is	 ?calculated	 ?as	 ?14	 ?days	 ?younger	 ?than	 ?the	 ?menstrual	 ?age	 ?(Sperber	 ?and	 ?Guttman,	 ?2010)	 ?.	 ?	 ?These	 ?dates	 ?are	 ?open	 ?to	 ?interpretation	 ?and	 ?rely	 ?on	 ?patients	 ?to	 ?correctly	 ?report	 ?the	 ?first	 ?day	 ?of	 ?their	 ?last	 ?menstrual	 ?cycle.	 ?	 ?To	 ?improve	 ?the	 ?accuracy	 ?of	 ?dating	 ?a	 ?human	 ?conceptus,	 ?other	 ?measurements	 ?may	 ?be	 ?utilized.	 ?	 ?	 ?Crown-??rump	 ?length	 ?is	 ?the	 ?earliest	 ?measurement	 ?that	 ?can	 ?accurately	 ?be	 ?used	 ?to	 ?date	 ?a	 ?developing	 ?embryo.	 ?Crown-??rump	 ?length	 ?can	 ?reliably	 ?be	 ?used	 ?on	 ?an	 ?embryo	 ?as	 ?young	 ?as	 ?6	 ?weeks.	 ?Crown-??rump	 ?length	 ?involves	 ?measuring	 ?the	 ?height	 ?of	 ?the	 ?fetus	 ?when	 ?it	 ?is	 ?in	 ?a	 ?passive	 ?C-??shape,	 ?not	 ?stretched	 ?out,	 ?from	 ?the	 ?tip	 ?of	 ?the	 ?head	 ?to	 ?the	 ?base	 ? 	 ? 2	 ?of	 ?the	 ?buttocks.	 ?	 ?When	 ?this	 ?method	 ?is	 ?being	 ?used	 ?on	 ?a	 ?live	 ?fetus	 ?via	 ?ultrasound,	 ?a	 ?best	 ?of	 ?three	 ?measurements	 ?should	 ?be	 ?taken.	 ?Maternal	 ?factors	 ?can	 ?alter	 ?the	 ?accuracy	 ?of	 ?the	 ?crown-??rump	 ?length,	 ?as	 ?maternal	 ?age,	 ?smoking	 ?and	 ?folic	 ?acid	 ?intake	 ?all	 ?play	 ?a	 ?role	 ?in	 ?the	 ?size	 ?of	 ?the	 ?fetus	 ?(Altman	 ?and	 ?Chitty,	 ?1997)	 ?.	 ?In	 ?fetuses	 ?aged	 ?13-??25	 ?weeks,	 ?femur	 ?length	 ?can	 ?also	 ?be	 ?used	 ?to	 ?estimate	 ?gestational	 ?age	 ?(Altman	 ?and	 ?Chitty,	 ?1997).	 ?	 ? Two	 ?methods	 ?based	 ?on	 ?the	 ?size	 ?of	 ?the	 ?cranium	 ?exist	 ?for	 ?dating	 ?human	 ?fetuses,	 ?these	 ?are	 ?head	 ?circumference	 ?and	 ?the	 ?biparietal	 ?diameter.	 ?	 ?Head	 ?circumference	 ?is	 ?a	 ?measurement	 ?independent	 ?of	 ?head	 ?shape,	 ?whereas	 ?the	 ?biparietal	 ?diameter	 ?is	 ?dependent	 ?on	 ?head	 ?shape,	 ?and	 ?requires	 ?the	 ?cephalic	 ?index	 ?to	 ?be	 ?used	 ?to	 ?account	 ?for	 ?a	 ?dolicocephalic	 ?versus	 ?brachycephalic	 ?head	 ?type.	 ?It	 ?has	 ?been	 ?recommended	 ?that	 ?due	 ?to	 ?the	 ?inaccuracy	 ?of	 ?the	 ?results,	 ?that	 ?the	 ?biparietal	 ?diameter	 ?be	 ?replaced	 ?with	 ?head	 ?circumference	 ?measurements	 ?(Altman	 ?and	 ?Chitty,	 ?1997;	 ?Hadlock	 ?et	 ?al.,	 ?1981).	 ?Head	 ?circumference	 ?is	 ?calculated	 ?by	 ?taking	 ?an	 ?anteroposterior	 ?and	 ?a	 ?biparietal	 ?measurement	 ?through	 ?a	 ?horizontal	 ?section	 ?of	 ?a	 ?fetal	 ?head	 ?at	 ?the	 ?level	 ?of	 ?the	 ?ventricles.	 ?	 ?Mathematical	 ?formulas	 ?are	 ?utilized	 ?to	 ?calculate	 ?the	 ?head	 ?circumference	 ?value,	 ?then	 ?this	 ?value	 ?is	 ?used	 ?in	 ?an	 ?additional	 ?formula	 ?to	 ?determine	 ?the	 ?gestational	 ?age	 ?(Altman	 ?and	 ?Chitty,	 ?1997).	 ?Streeter	 ?was	 ?one	 ?of	 ?the	 ?first	 ?individuals	 ?to	 ?attempt	 ?to	 ?quantify	 ?gestational	 ?age	 ?of	 ?embryos	 ?in	 ?the	 ?early	 ?1900?s,	 ?based	 ?on	 ?measured	 ?values	 ?such	 ?as	 ?weight,	 ?head	 ?size	 ?and	 ?foot	 ?length	 ?(Streeter,	 ?1920)	 ?.	 ?	 ?These	 ?collections	 ?he	 ?viewed	 ?would	 ?ultimately	 ?become	 ?part	 ?of	 ?the	 ?Carnegie	 ?Collection,	 ?an	 ?atlas	 ?by	 ?which	 ?embryologic	 ?development	 ?is	 ?based	 ?(O'Rahilly	 ?and	 ?Muller,	 ?2010).	 ?	 ?Another	 ?investigator	 ?(Hern,	 ?1984)	 ?analyzed	 ?1800	 ?tissue	 ?specimens	 ?ranging	 ?from	 ?10-??24	 ?weeks	 ?obtained	 ?post-??abortion	 ?and	 ?measured	 ?weight,	 ?knee	 ?to	 ?heel	 ?length,	 ?biparietal	 ?diameter,	 ?placental	 ?weight	 ?and	 ? 	 ? 3	 ?amniotic	 ?fluid	 ?volume.	 ?He	 ?preferred	 ?foot	 ?length	 ?as	 ?a	 ?guide,	 ?as	 ?this	 ?value	 ?is	 ?easily	 ?obtainable	 ?and	 ?is	 ?not	 ?subject	 ?to	 ?significant	 ?operator	 ?error.	 ?	 ?Most	 ?recently,	 ?O?Rahilly	 ?and	 ?Muller	 ?re-??evaluated	 ?the	 ?staging	 ?of	 ?the	 ?Carnegie	 ?collection	 ?(O'Rahilly	 ?and	 ?Muller,	 ?2010).	 ?They	 ?commented	 ?that	 ?crown-??rump	 ?lengths	 ?should	 ?be	 ?used	 ?with	 ?caution,	 ?as	 ?this	 ?is	 ?difficult	 ?values	 ?to	 ?obtain	 ?due	 ?to	 ?arbitrary	 ?landmarks,	 ?and	 ?instead,	 ?the	 ?greatest	 ?length	 ?of	 ?the	 ?specimen	 ?(excluding	 ?the	 ?lower	 ?limbs)	 ?should	 ?be	 ?used.	 ?In	 ?general	 ?female	 ?embryos	 ?and	 ?fetuses	 ?will	 ?be	 ?smaller	 ?and	 ?shorter	 ?in	 ?length	 ?than	 ?male	 ?embryos.	 ?The	 ?specimens	 ?in	 ?my	 ?study	 ?have	 ?had	 ?their	 ?ages	 ?determined	 ?by	 ?a	 ?combination	 ?of	 ?last	 ?reported	 ?menstrual	 ?period,	 ?crown-??rump	 ?length	 ?and	 ?foot	 ?size.	 ?	 ?1.1. Development of the primary palate 	 ? Primary	 ?palate	 ?or	 ?lip	 ?formation	 ?begins	 ?during	 ?the	 ?4th	 ?week	 ?of	 ?human	 ?development.	 ?The	 ?neural	 ?crest	 ?cells	 ?begin	 ?to	 ?migrate	 ?away	 ?from	 ?the	 ?neural	 ?tube	 ?into	 ?the	 ?region	 ?of	 ?the	 ?developing	 ?face	 ?at	 ?21	 ?days	 ?gestation	 ?(O'Rahilly	 ?and	 ?Muller,	 ?2007;	 ?Sperber	 ?and	 ?Guttman,	 ?2010).	 ?Shortly	 ?after,	 ?by	 ?24	 ?days,	 ?the	 ?frontonasal	 ?process,	 ?and	 ?the	 ?maxillary	 ?and	 ?mandibular	 ?processes	 ?from	 ?the	 ?first	 ?arch	 ?have	 ?formed	 ?and	 ?have	 ?surrounded	 ?the	 ?primitive	 ?stomodeum	 ?(Mossey	 ?et	 ?al.,	 ?2009;	 ?Sperber	 ?and	 ?Guttman,	 ?2010).	 ?Initially	 ?there	 ?is	 ?a	 ?buccopharyngeal	 ?membrane	 ?separating	 ?the	 ?oral	 ?cavity	 ?from	 ?the	 ?developing	 ?gastrointestinal	 ?tract.	 ?	 ?The	 ?epithelial	 ?lining	 ?of	 ?all	 ?of	 ?the	 ?structures	 ?anterior	 ?to	 ?the	 ?membrane	 ?is	 ?of	 ?ectodermal	 ?origin,	 ?and	 ?the	 ?lining	 ?of	 ?everything	 ?posterior	 ?to	 ?the	 ?membrane	 ?is	 ?thought	 ?to	 ?be	 ?of	 ?endodermal	 ?origin.	 ?	 ?The	 ?position	 ?of	 ?the	 ?precise	 ?line	 ?dividing	 ?ectodermal	 ?and	 ?endodermal	 ?epithelium	 ?in	 ?the	 ? 	 ? 4	 ?oral	 ?cavity	 ?is	 ?not	 ?known.	 ?As	 ?early	 ?as	 ?28	 ?days	 ?the	 ?buccopharyngeal	 ?membrane	 ?ruptures,	 ?connecting	 ?the	 ?two	 ?cavities	 ?(Sadler	 ?and	 ?Langman,	 ?2010a;	 ?Sperber	 ?and	 ?Guttman,	 ?2010).	 ?The	 ?next	 ?major	 ?feature	 ?of	 ?the	 ?face	 ?to	 ?form	 ?is	 ?the	 ?nose.	 ?Initially	 ?nasal	 ?placodes	 ?form	 ?as	 ?ectodermal	 ?thickenings	 ?on	 ?the	 ?frontonasal	 ?process	 ?and	 ?by	 ?33	 ?days	 ?the	 ?placodes	 ?have	 ?invaginated	 ?into	 ?the	 ?underlying	 ?mesenchyme	 ?to	 ?become	 ?nasal	 ?pits.	 ?The	 ?medial	 ?nasal	 ?prominences	 ?are	 ?medial	 ?to	 ?the	 ?nasal	 ?pits	 ?while	 ?the	 ?lateral	 ?nasal	 ?prominences	 ?are	 ?lateral	 ?(Sadler	 ?and	 ?Langman,	 ?2010a;	 ?Sperber	 ?and	 ?Guttman,	 ?2010).	 ?	 ?On	 ?each	 ?side	 ?of	 ?the	 ?stomodeum,	 ?the	 ?maxillary	 ?prominences	 ?bud	 ?outwards.	 ?The	 ?inferior	 ?mandibular	 ?prominences	 ?complete	 ?the	 ?circle	 ?of	 ?facial	 ?prominences	 ?that	 ?ring	 ?the	 ?stomodeum.	 ?The	 ?fate	 ?of	 ?each	 ?prominence	 ?is	 ?distinct.	 ?The	 ?medial	 ?nasal	 ?prominences	 ?contribute	 ?to	 ?the	 ?facial	 ?midline,	 ?nasal	 ?septum,	 ?centre	 ?of	 ?the	 ?nose,	 ?philtrum,	 ?premaxilla	 ?and	 ?4	 ?incisors.	 ?The	 ?lateral	 ?nasal	 ?prominences	 ?contribute	 ?to	 ?the	 ?nasal	 ?turbinates	 ?as	 ?shown	 ?in	 ?chicken	 ?experiments	 ?(MacDonald	 ?et	 ?al.,	 ?2004).	 ?The	 ?maxillary	 ?prominences	 ?form	 ?the	 ?bones	 ?of	 ?the	 ?palate	 ?and	 ?upper	 ?jaw	 ?(Lee	 ?et	 ?al.,	 ?2004)	 ?as	 ?well	 ?as	 ?all	 ?teeth	 ?posterior	 ?to	 ?the	 ?lateral	 ?incisors.	 ?The	 ?mandibular	 ?prominences	 ?form	 ?the	 ?entire	 ?mandible,	 ?parts	 ?of	 ?the	 ?temporomandibular	 ?joint	 ?as	 ?well	 ?as	 ?ossicles	 ?in	 ?the	 ?inner	 ?ear.	 ?As	 ?these	 ?facial	 ?prominences	 ?mature,	 ?there	 ?are	 ?two	 ?developmental	 ?processes	 ?bringing	 ?the	 ?facial	 ?prominences	 ?together,	 ?merging	 ?and	 ?fusion.	 ?Merging	 ?involves	 ?removal	 ?of	 ?a	 ?groove	 ?or	 ?furrow	 ?between	 ?partially	 ?attached	 ?developmental	 ?structures	 ?by	 ?the	 ?migration	 ?and	 ?proliferation	 ?of	 ?underlying	 ?mesenchymal	 ?cells	 ?to	 ?fill	 ?in	 ?these	 ?grooves	 ?(Sperber	 ?and	 ?Guttman,	 ?2010).	 ?Fusion,	 ?on	 ?the	 ?other	 ?hand,	 ?occurs	 ?when	 ?two	 ?epithelial-??lined	 ?structures	 ?contact	 ?and	 ?create	 ?a	 ?bilayered	 ?epithelial	 ?seam,	 ?which	 ?is	 ? 	 ? 5	 ?thought	 ?to	 ?be	 ?removed	 ?by	 ?apoptosis,	 ?epithelial	 ?to	 ?mesenchymal	 ?cell	 ?transformation,	 ?and	 ?cell	 ?migration	 ?to	 ?adjacent	 ?epithelia.	 ?Most	 ?of	 ?the	 ?work	 ?on	 ?fusion	 ?in	 ?the	 ?face	 ?was	 ?carried	 ?out	 ?on	 ?the	 ?secondary	 ?palate	 ?(Fitchett	 ?and	 ?Hay,	 ?1989;	 ?Shuler,	 ?1995;	 ?Sperber	 ?and	 ?Guttman,	 ?2010)	 ?	 ?so	 ?it	 ?is	 ?not	 ?clear	 ?whether	 ?all	 ?of	 ?the	 ?aforementioned	 ?mechanisms	 ?are	 ?at	 ?play	 ?during	 ?primary	 ?palate	 ?formation.	 ?The	 ?only	 ?data	 ?on	 ?EMT	 ?in	 ?the	 ?primary	 ?palate	 ?comes	 ?from	 ?the	 ?chicken	 ?embryo	 ?in	 ?which	 ?the	 ?seam	 ?between	 ?the	 ?frontonasal	 ?mass	 ?and	 ?maxillary	 ?prominence	 ?was	 ?seen	 ?to	 ?transform	 ?to	 ?mesenchyme	 ?(Sun	 ?et	 ?al.,	 ?2000).	 ?	 ?In	 ?the	 ?mouse	 ?model,	 ?there	 ?is	 ?typically	 ?apoptosis	 ?at	 ?the	 ?point	 ?of	 ?contact	 ?of	 ?the	 ?medial	 ?nasal	 ?and	 ?maxillary	 ?prominences	 ?(Jiang	 ?et	 ?al.,	 ?2006).	 ?Fusion	 ?in	 ?the	 ?primary	 ?palate	 ?occurs	 ?in	 ?41	 ?day	 ?embryos	 ?when	 ?the	 ?maxillary	 ?prominences	 ?contact	 ?the	 ?medial	 ?and	 ?lateral	 ?nasal	 ?processes	 ?(Sadler	 ?and	 ?Langman,	 ?2010a;	 ?Sperber	 ?and	 ?Guttman,	 ?2010).	 ?	 ?The	 ?point	 ?of	 ?contact	 ?between	 ?the	 ?maxillary,	 ?medial	 ?and	 ?lateral	 ?nasal	 ?prominences	 ?is	 ?the	 ?area	 ?most	 ?susceptible	 ?to	 ?clefting.	 ?Merging	 ?takes	 ?place	 ?in	 ?the	 ?paired	 ?medial	 ?nasal	 ?processes,	 ?and	 ?a	 ?result	 ?of	 ?this	 ?process,	 ?the	 ?intermaxillary	 ?segment	 ?is	 ?formed.	 ?	 ?Two	 ?other	 ?places	 ?in	 ?the	 ?face	 ?undergo	 ?merging,	 ?The	 ?lower	 ?jaw	 ?also	 ?develops	 ?by	 ?merging	 ?of	 ?the	 ?midline	 ?of	 ?the	 ?mandibular	 ?prominences	 ?in	 ?a	 ?posterior	 ?to	 ?anterior	 ?fashion	 ?(Oostrom	 ?et	 ?al.,	 ?1996).	 ?Finally,	 ?the	 ?maxillary	 ?and	 ?lateral	 ?nasal	 ?processes	 ?are	 ?separated	 ?by	 ?a	 ?deep	 ?furrow	 ?that	 ?fills	 ?in	 ?by	 ?merging.	 ?Failure	 ?of	 ?merging	 ?in	 ?this	 ?region	 ?leads	 ?to	 ?an	 ?exposed	 ?nasolacrimal	 ?duct	 ?.	 ?	 ?Thus	 ?by	 ?the	 ?end	 ?of	 ?47	 ?days,	 ?the	 ?primary	 ?palate	 ?has	 ?completed	 ?development	 ?and	 ?residual	 ?grooves	 ?are	 ?being	 ?filled	 ?in	 ?by	 ?merging.	 ?	 ?	 ? 	 ? 6	 ?1.2. Secondary palate development   1.3.1 Embryonic period  	 ?During	 ?the	 ?6th	 ?week	 ?of	 ?gestation,	 ?the	 ?secondary	 ?palate	 ?begins	 ?to	 ?develop	 ?(Dixon	 ?et	 ?al.,	 ?2011).	 ?It	 ?starts	 ?initially	 ?as	 ?two	 ?outgrowths	 ?from	 ?the	 ?maxillary	 ?prominences	 ?which	 ?are	 ?oriented	 ?in	 ?a	 ?vertical	 ?direction	 ?and	 ?lay	 ?on	 ?either	 ?side	 ?of	 ?the	 ?tongue	 ?(Greene	 ?and	 ?Pisano,	 ?2010;	 ?Sadler	 ?and	 ?Langman,	 ?2010a).	 ?	 ?	 ?During	 ?the	 ?8th	 ?week	 ?of	 ?development,	 ?the	 ?palatal	 ?shelves	 ?reorient	 ?in	 ?to	 ?a	 ?horizontal	 ?position	 ?over	 ?in	 ?just	 ?a	 ?few	 ?hours	 ?(Bush	 ?and	 ?Jiang,	 ?2012;	 ?Gritli-??Linde,	 ?2007;	 ?Sadler	 ?and	 ?Langman,	 ?2010a;	 ?Sperber	 ?and	 ?Guttman,	 ?2010).	 ?While	 ?reorientation	 ?is	 ?occurring,	 ?the	 ?mandible	 ?is	 ?elongating	 ?and	 ?the	 ?fetal	 ?head	 ?tilts	 ?upwards.	 ?Both	 ?of	 ?these	 ?events	 ?help	 ?move	 ?the	 ?tongue	 ?out	 ?from	 ?between	 ?the	 ?shelves	 ?(Diewert,	 ?1985;	 ?Diewert,	 ?1986).	 ?	 ?The	 ?palatal	 ?shelves	 ?continue	 ?to	 ?grow	 ?towards	 ?each	 ?other	 ?and	 ?contact	 ?at	 ?the	 ?medial	 ?edge	 ?epithelia.	 ?A	 ?transient	 ?bilayered	 ?epithelium	 ?or	 ?midline	 ?epithelial	 ?seam	 ?(mes)	 ?is	 ?formed	 ?at	 ?this	 ?time	 ?(Gritli-??Linde,	 ?2007;	 ?Mossey	 ?et	 ?al.,	 ?2009).	 ?This	 ?process	 ?of	 ?fusion	 ?requires	 ?adherence	 ?of	 ?the	 ?two	 ?epithelia,	 ?which	 ?occurs	 ?via	 ?cell	 ?adhesion	 ?molecules	 ?and	 ?desmosomes	 ?(Mogass	 ?et	 ?al.,	 ?2000;	 ?Mossey	 ?et	 ?al.,	 ?2009).	 ?	 ?Growth	 ?factors	 ?are	 ?also	 ?important	 ?in	 ?palatal	 ?fusion	 ?as	 ?well.	 ?Transforming	 ?growth	 ?factor	 ??	 ?(TGF?),	 ?epidermal	 ?growth	 ?factor	 ?receptor	 ?(EGFR)	 ?and	 ?members	 ?of	 ?the	 ?transforming	 ?growth	 ?factor	 ??	 ?superfamily	 ?(TGF?)	 ?have	 ?all	 ?been	 ?shown	 ?to	 ?play	 ?a	 ?role	 ?in	 ?fusion	 ?of	 ?the	 ?palatal	 ?shelves	 ?(Cui	 ?et	 ?al.,	 ?2003;	 ?Cui	 ?et	 ?al.,	 ?2005;	 ?Dudas	 ?et	 ?al.,	 ?2004;	 ?Kaartinen	 ?et	 ?al.,	 ?1997;	 ?Kaartinen	 ?et	 ?al.,	 ?1995;	 ?Miettinen	 ?et	 ?al.,	 ?1999;	 ?Proetzel	 ?et	 ?al.,	 ?1995).	 ?	 ?After	 ?the	 ?palatal	 ? 	 ? 7	 ?shelves	 ?have	 ?contacted	 ?and	 ?fused,	 ?the	 ?midline	 ?epithelial	 ?seam	 ?has	 ?to	 ?be	 ?removed	 ?to	 ?create	 ?an	 ?intact	 ?palate.	 ?This	 ?is	 ?done	 ?by	 ?a	 ?number	 ?of	 ?different	 ?mechanisms	 ?including	 ?apoptosis,	 ?epithelial	 ?to	 ?mesenchymal	 ?transformation	 ?and	 ?migration	 ?of	 ?the	 ?epithelial	 ?cells	 ?to	 ?adjacent	 ?epithelia.	 ?	 ?TUNEL	 ?(terminal	 ?deoxynucleotidyl	 ?transferase	 ?nick-??end	 ?labeling)	 ?assays	 ?have	 ?identified	 ?the	 ?importance	 ?of	 ?apoptosis	 ?in	 ?the	 ?removal	 ?of	 ?the	 ?midline	 ?seam	 ?(Cuervo	 ?et	 ?al.,	 ?2002;	 ?Nawshad,	 ?2008).	 ?	 ?The	 ?role	 ?of	 ?epithelial-??mesenchymal	 ?transformation	 ?is	 ?controversial	 ?(Fitchett	 ?and	 ?Hay,	 ?1989;	 ?Nawshad,	 ?2008).	 ?Some	 ?believe	 ?that	 ?the	 ?epithelial	 ?cells	 ?are	 ?not	 ?transforming,	 ?but	 ?are	 ?instead	 ?migrating	 ?to	 ?the	 ?adjacent	 ?epithelia	 ?(Cuervo	 ?and	 ?Covarrubias,	 ?2004;	 ?Xu	 ?et	 ?al.,	 ?2006).	 ?The	 ?newer	 ?genetic	 ?methods	 ?of	 ?tracing	 ?epithelial	 ?cells	 ?are	 ?better	 ?and	 ?on	 ?occasion	 ?it	 ?is	 ?possible	 ?to	 ?see	 ?labeled	 ?epithelial	 ?cells	 ?in	 ?the	 ?mesenchyme	 ?(Jin	 ?and	 ?Ding,	 ?2006).	 ?Regardless	 ?of	 ?the	 ?mechanism,	 ?the	 ?secondary	 ?palate	 ?fusion	 ?results	 ?in	 ?removal	 ?of	 ?the	 ?epithelial	 ?seam	 ?and	 ?as	 ?a	 ?result	 ?sometimes	 ?epithelial	 ?islands	 ?remain	 ?in	 ?the	 ?midline.	 ?These	 ?epithelial	 ?remnants	 ?are	 ?thought	 ?to	 ?be	 ?the	 ?cause	 ?of	 ?midline	 ?cysts	 ?otherwise	 ?known	 ?as	 ?Bohn?s	 ?nodules	 ?(Monteleone	 ?and	 ?McLellan,	 ?1964;	 ?Saunders,	 ?1972).	 ?The	 ?final	 ?events	 ?of	 ?fusion	 ?connect	 ?the	 ?nasal	 ?side	 ?of	 ?the	 ?secondary	 ?palate	 ?to	 ?the	 ?nasal	 ?septum	 ?and	 ?the	 ?anteriorly	 ?the	 ?palatal	 ?shelves	 ?join	 ?with	 ?the	 ?primary	 ?palate.	 ?Failure	 ?of	 ?one	 ?of	 ?the	 ?palatal	 ?shelves	 ?to	 ?fuse	 ?with	 ?the	 ?nasal	 ?septum	 ?causes	 ?unilateral	 ?cleft	 ?palate	 ?whereas	 ?failure	 ?to	 ?fuse	 ?on	 ?both	 ?sides	 ?of	 ?the	 ?septum	 ?causes	 ?bilateral	 ?cleft	 ?palate	 ?(Dixon	 ?et	 ?al.,	 ?2011)	 ?	 ?	 ?	 ? 	 ? 8	 ?1.3.2 Development of the hard palate in the fetal period 	 ?The	 ?hard	 ?palate	 ?is	 ?comprised	 ?of	 ?the	 ?primary	 ?palate	 ?and	 ?the	 ?anterior	 ?aspect	 ?of	 ?the	 ?secondary	 ?palate	 ?that	 ?is	 ?supported	 ?by	 ?bone.	 ?The	 ?bones	 ?of	 ?the	 ?hard	 ?palate	 ?consist	 ?of	 ?paired	 ?palatine	 ?process	 ?of	 ?the	 ?maxillary	 ?bone	 ?and	 ?more	 ?posteriorly,	 ?the	 ?palatine	 ?bones.	 ?These	 ?bones	 ?develop	 ?from	 ?intramembranous	 ?ossification,	 ?differentiating	 ?directly	 ?from	 ?neural	 ?crest	 ?mesenchyme	 ?to	 ?osteoblasts.	 ?	 ?The	 ?palatine	 ?processes	 ?of	 ?the	 ?maxilla	 ?form	 ?from	 ?de	 ?novo	 ?ossification	 ?centers	 ?that	 ?are	 ?initially	 ?separate	 ?from	 ?the	 ?maxillary	 ?bone	 ?(Bush	 ?and	 ?Jiang,	 ?2012).	 ?The	 ?palatal	 ?processes	 ?of	 ?the	 ?palatine	 ?bones	 ?form	 ?by	 ?expansion	 ?of	 ?osteogenic	 ?fronts	 ?from	 ?the	 ?lateral	 ?aspects	 ?of	 ?the	 ?bone	 ?towards	 ?the	 ?midline	 ?(Baek	 ?et	 ?al.,	 ?2011;	 ?Bush	 ?and	 ?Jiang,	 ?2012).	 ?	 ?When	 ?defects	 ?in	 ?the	 ?bony	 ?hard	 ?palate	 ?develop	 ?after	 ?successful	 ?soft	 ?tissue	 ?fusion,	 ?a	 ?form	 ?of	 ?submucous	 ?cleft	 ?of	 ?the	 ?palate	 ?occurs.	 ?	 ?	 ?	 ?1.3.3 Development of the soft palate in the fetal period 	 ?The	 ?soft	 ?palate	 ?is	 ?the	 ?posterior	 ?muscular	 ?portion	 ?of	 ?the	 ?secondary	 ?palate.	 ?	 ?It	 ?has	 ?critical	 ?functions	 ?during	 ?swallowing	 ?and	 ?speech.	 ?Dysfunction	 ?in	 ?soft	 ?palate	 ?movement	 ?or	 ?ability	 ?or	 ?create	 ?a	 ?seal	 ?with	 ?the	 ?pharynx	 ?leads	 ?to	 ?velopharyngeal	 ?insufficiency,	 ?which	 ?is	 ?characterized	 ?by	 ?nasal	 ?regurgitation	 ?during	 ?swallowing,	 ?hypernasal	 ?speech,	 ?and	 ?in	 ?very	 ?severe	 ?situations,	 ?failure	 ?to	 ?thrive	 ?(Ha	 ?et	 ?al.,	 ?2013;	 ?Itani	 ?et	 ?al.,	 ?2000).	 ?	 ? 	 ? 9	 ?Soft	 ?palate	 ?development	 ?in	 ?humans	 ?begins	 ?after	 ?palatogenesis	 ?of	 ?the	 ?hard	 ?palate,	 ?and	 ?is	 ?generally	 ?thought	 ?to	 ?occur	 ?during	 ?weeks	 ?9-??12	 ?of	 ?development.	 ?	 ?By	 ?16-??17	 ?weeks,	 ?soft	 ?palate	 ?myogenesis	 ?is	 ?also	 ?complete	 ?and	 ?this	 ?is	 ?considered	 ?to	 ?be	 ?the	 ?end	 ?of	 ?palate	 ?morphogenesis	 ?(Cohen	 ?et	 ?al.,	 ?1993).	 ?Significant	 ?research	 ?has	 ?gone	 ?towards	 ?development	 ?of	 ?the	 ?hard	 ?palate,	 ?but	 ?relatively	 ?little	 ?attention	 ?has	 ?been	 ?given	 ?to	 ?the	 ?mechanism	 ?of	 ?soft	 ?palate	 ?development.	 ?	 ?The	 ?landmark	 ?study	 ?by	 ?which	 ?most	 ?craniofacial	 ?textbooks	 ?cite	 ?the	 ?mechanism	 ?of	 ?soft	 ?palate	 ?development	 ?is	 ?from	 ?Burdi	 ?and	 ?Faist	 ?(Burdi	 ?and	 ?Faist,	 ?1967).	 ?	 ?Their	 ?hypothesis	 ?is	 ?that	 ?after	 ?the	 ?hard	 ?palate	 ?develops	 ?by	 ?fusion	 ?of	 ?the	 ?palatal	 ?shelves,	 ?the	 ?soft	 ?palate	 ?forms	 ?through	 ?migration	 ?and	 ?proliferation	 ?of	 ?two	 ?subepithelial	 ?mesenchymal	 ?growth	 ?centers	 ?at	 ?the	 ?posterior	 ?edge	 ?of	 ?the	 ?newly	 ?fused	 ?hard	 ?palate,	 ?with	 ?groove	 ?between	 ?these	 ?structures	 ?filled	 ?in	 ?via	 ?merging	 ?(Fig.	 ?1.1).	 ?Thus	 ?in	 ?the	 ?Burdi	 ?and	 ?Faist	 ?mechanism	 ?of	 ?soft	 ?palate	 ?development,	 ?only	 ?merging	 ?occurs	 ?and	 ?	 ?there	 ?would	 ?be	 ?no	 ?midline	 ?seam	 ?between	 ?the	 ?soft	 ?palate	 ?shelves.	 ?	 ?Their	 ?study	 ?had	 ?31	 ?embryos	 ?and	 ?fetuses,	 ?ranging	 ?in	 ?age	 ?from	 ?7-??12	 ?weeks	 ?and	 ?in	 ?size	 ?from	 ?18-??75mm	 ?crown-??rump	 ?length	 ?(Table	 ?3.2).	 ?	 ?Of	 ?these	 ?specimens,	 ?ten	 ?were	 ?of	 ?the	 ?age	 ?where	 ?fusion	 ?of	 ?the	 ?hard	 ?palate	 ?would	 ?be	 ?complete,	 ?but	 ?only	 ?two	 ?were	 ?at	 ?an	 ?age	 ?where	 ?soft	 ?palate	 ?shelves	 ?are	 ?likely	 ?to	 ?have	 ?contacted	 ?but	 ?not	 ?yet	 ?fused	 ?(Table	 ?3.2).	 ?	 ?With	 ?such	 ?a	 ?low	 ?sample	 ?size,	 ?it	 ?is	 ?not	 ?surprising	 ?that	 ?in	 ?their	 ?observations,	 ?no	 ?seam	 ?or	 ?epithelial	 ?remnants	 ?were	 ?seem	 ?in	 ?the	 ?soft	 ?palate.	 ?Furthermore,	 ?others	 ?have	 ?reported	 ?that	 ?the	 ?seam	 ?in	 ?the	 ?soft	 ?palate	 ?is	 ?subject	 ?to	 ?rapid	 ?degradation	 ?(Kitamura,	 ?1966).	 ?	 ?Poswillo (Poswillo, 1974) also proposed a similar mechanism of soft palate development to that of Burdi and Faist  in which fusion occurs in the anterior two thirds of the soft palate while  	 ? 10	 ?the posterior third including uvula, develops by merging. With	 ?the	 ?limited	 ?sample	 ?size	 ?in	 ?the	 ?Burdi	 ?study,	 ?it	 ?is	 ?clear	 ?that	 ?additional	 ?investigations	 ?are	 ?necessary	 ?to	 ?determine	 ?whether	 ?or	 ?not	 ?the	 ?soft	 ?palate	 ?forms	 ?by	 ?fusion	 ?or	 ?merging.	 ?	 ?	 ?Figure	 ?1.1.	 ?Schematic	 ?of	 ?palate	 ?closure	 ?from	 ?Burdi	 ?and	 ?Faist,	 ?1967.	 ?	 ?There	 ?are	 ?several	 ?additional	 ?studies	 ?that	 ?have	 ?looked	 ?specifically	 ?at	 ?the	 ?soft	 ?palate.	 ?In	 ?one	 ?study	 ?human	 ?specimens	 ?age	 ?7-??11	 ?weeks	 ?(Wood	 ?and	 ?Kraus,	 ?1962)	 ?were	 ?examined.	 ?In	 ?the	 ?text	 ?of	 ?the	 ?article,	 ?these	 ?authors	 ?indicated	 ?that	 ?the	 ?soft	 ?palate	 ?shelves	 ?fused	 ?but	 ?did	 ?not	 ?illustrate	 ?the	 ?seam.	 ?They	 ?directed	 ?most	 ?of	 ?their	 ?attention	 ?A) The primary palate grows posteriorly to meet the palatal shelves. B) Open arrows show areas of fusion with epithelial seams whereas stippled black arrow shows mesenchymal proliferation and merging. C) Continued merging of the soft palate due to mesenchymal proliferation shown by stippled black arrow. D) Fusion is still continuing anteriorly but solid black arrows show that uvula is forming via merging.   	 ? 11	 ?to	 ?the	 ?hard	 ?palate	 ?where	 ?they	 ?observed	 ?numerous	 ?keratinized	 ?epithelial	 ?inclusion	 ?bodies,	 ?or	 ?pearls.	 ?In	 ?contrast	 ?in	 ?the	 ?soft	 ?palate	 ?no	 ?such	 ?islands	 ?of	 ?epithelium	 ?were	 ?seen.	 ?It	 ?is	 ?worth	 ?noting	 ?that	 ?the	 ?epithelium	 ?of	 ?the	 ?hard	 ?palate	 ?may	 ?have	 ?a	 ?different,	 ?ectodermal	 ?origin	 ?than	 ?that	 ?of	 ?the	 ?soft	 ?palate	 ?which	 ?is	 ?most	 ?likely	 ?derived	 ?from	 ?endoderm.	 ?The	 ?difference	 ?in	 ?origins	 ?could	 ?explain	 ?why	 ?no	 ?seam	 ?or	 ?epithelial	 ?islands	 ?were	 ?observed	 ?in	 ?the	 ?soft	 ?palate.	 ?(Sperber	 ?and	 ?Guttman,	 ?2010).	 ?	 ?In	 ?a	 ?Japanese	 ?study,	 ?epithelial	 ?remnants	 ?have	 ?been	 ?located	 ?in	 ?the	 ?midline	 ?of	 ?the	 ?soft	 ?palate	 ?of	 ?specimens	 ?between	 ?53-??55	 ?days	 ?post-??conception	 ?but	 ?had	 ?disappeared	 ?by	 ?60	 ?days	 ?(Kitamura,	 ?1966).	 ?	 ?Palatal	 ?cysts	 ?of	 ?the	 ?newborn	 ?located	 ?in	 ?the	 ?hard	 ?palate	 ?are	 ?very	 ?common	 ?(54-??79%	 ?of	 ?newborns	 ?(Monteagudo	 ?et	 ?al.,	 ?2012;	 ?Paula	 ?et	 ?al.,	 ?2006).	 ?By	 ?comparison,	 ?midline	 ?palatal	 ?cysts	 ?of	 ?the	 ?soft	 ?palate	 ?are	 ?extremely	 ?uncommon	 ?.	 ?Only	 ?six	 ?case	 ?reports	 ?of	 ?epidermoid	 ?cysts	 ?requiring	 ?surgical	 ?intervention	 ?in	 ?the	 ?soft	 ?palate	 ?have	 ?been	 ?reported	 ?as	 ?of	 ?2010	 ?(Suga	 ?et	 ?al.,	 ?2010).	 ?	 ?	 ? The	 ?main	 ?opponent	 ?of	 ?the	 ?Burdi	 ?work	 ?was	 ?Smiley	 ?(Smiley,	 ?1972;	 ?Smiley,	 ?1975;	 ?Smiley	 ?and	 ?Koch,	 ?1975;	 ?Suga	 ?et	 ?al.,	 ?2010).	 ?He	 ?strongly	 ?believed	 ?that	 ?fusion	 ?was	 ?the	 ?main	 ?mechanism	 ?of	 ?soft	 ?palate	 ?development.	 ?He	 ?questioned	 ?how	 ?certain	 ?types	 ?of	 ?submucous	 ?clefts	 ?could	 ?develop	 ?if	 ?merging	 ?was	 ?the	 ?only	 ?process	 ?occurring,	 ?for	 ?example	 ?a	 ?perforation	 ?in	 ?the	 ?posterior	 ?hard	 ?and	 ?anterior	 ?soft	 ?palate,	 ?with	 ?intact	 ?soft	 ?palate	 ?posterior	 ?to	 ?the	 ?cleft	 ?defect	 ?(Smiley,	 ?1972).	 ?He	 ?also	 ?believed	 ?that	 ?differential	 ?rates	 ?of	 ?midline	 ?epithelium	 ?degradation	 ?exist,	 ?with	 ?the	 ?soft	 ?palate	 ?seam	 ?forming	 ?later	 ?but	 ?degrading	 ?much	 ?more	 ?rapidly	 ?than	 ?the	 ?hard	 ?palate	 ?seam	 ?(Mato	 ?et	 ?al.,	 ?1972).	 ?	 ?	 ? 	 ? 	 ? 12	 ?1.3.4 Muscle development in the soft palate 	 ?After	 ?seam	 ?formation	 ?and	 ?degradation	 ?the	 ?next	 ?critical	 ?event	 ?during	 ?soft	 ?palate	 ?morphogenesis	 ?is	 ?the	 ?invasion	 ?of	 ?myoblasts	 ?and	 ?formation	 ?of	 ?the	 ?palatine	 ?musculature.	 ?The	 ?palatine	 ?muscles	 ?develop	 ?from	 ?the	 ?first	 ?and	 ?fourth	 ?pharyngeal	 ?arches.	 ?The	 ?tensor	 ?veli	 ?palatini	 ?is	 ?the	 ?first	 ?palatal	 ?muscle	 ?to	 ?develop	 ?and	 ?the	 ?only	 ?one	 ?to	 ?develop	 ?from	 ?the	 ?first	 ?arch,	 ?and	 ?this	 ?is	 ?the	 ?only	 ?one	 ?to	 ?have	 ?innervation	 ?from	 ?the	 ?trigeminal	 ?nerve.	 ?	 ?	 ?It	 ?is	 ?composed	 ?of	 ?two	 ?heads,	 ?the	 ?lateral	 ?head	 ?originates	 ?on	 ?the	 ?sphenoid	 ?bone	 ?and	 ?the	 ?medial	 ?head	 ?originates	 ?at	 ?the	 ?auditory	 ?tube	 ?(De	 ?la	 ?Cuadra	 ?Blanco	 ?et	 ?al.,	 ?2012;	 ?Sperber	 ?and	 ?Guttman,	 ?2010).	 ?	 ?When	 ?fully	 ?developed,	 ?the	 ?two	 ?heads	 ?join	 ?and	 ?wrap	 ?around	 ?the	 ?pterygoid	 ?hamulus,	 ?inserting	 ?in	 ?to	 ?the	 ?palatine	 ?aponeurosis	 ?(De	 ?la	 ?Cuadra	 ?Blanco	 ?et	 ?al.,	 ?2012).	 ?The	 ?palatine	 ?aponeurosis	 ?is	 ?a	 ?fibrous	 ?band	 ?extending	 ?off	 ?the	 ?posterior	 ?of	 ?the	 ?hard	 ?palate.	 ?It	 ?serves	 ?as	 ?point	 ?of	 ?origin	 ?and	 ?insertion	 ?for	 ?the	 ?muscles	 ?of	 ?the	 ?soft	 ?palate.	 ?	 ?For	 ?the	 ?purpose	 ?of	 ?this	 ?study,	 ?continuity	 ?of	 ?aponeurosis	 ?and	 ?tensor	 ?veli	 ?palatini	 ?can	 ?serve	 ?as	 ?a	 ?marker	 ?for	 ?completion	 ?of	 ?soft	 ?palate	 ?fusion	 ?(De	 ?la	 ?Cuadra	 ?Blanco	 ?et	 ?al.,	 ?2012).	 ?	 ?	 ?	 ? The	 ?levator	 ?veli	 ?palatini,	 ?palatopharyngeus,	 ?musculus	 ?uvulus	 ?and	 ?palatoglossus	 ?muscles	 ?are	 ?all	 ?derived	 ?from	 ?the	 ?fourth	 ?pharyngeal	 ?arch	 ?and	 ?are	 ?innervated	 ?by	 ?the	 ?vagus	 ?nerve	 ?(Sperber	 ?and	 ?Guttman,	 ?2010).	 ?They	 ?all	 ?develop	 ?at	 ?approximately	 ?the	 ?same	 ?time	 ?in	 ?a	 ?fetus.	 ?	 ?Failure	 ?of	 ?any	 ?of	 ?these	 ?muscles	 ?to	 ?fully	 ?develop	 ?or	 ?reach	 ?its	 ?site	 ?of	 ?insertion	 ?can	 ?result	 ?in	 ?a	 ?cleft	 ?of	 ?the	 ?muscles,	 ?which	 ?is	 ?a	 ?variant	 ?of	 ?a	 ?submucous	 ?cleft.	 ?	 ?Because	 ?of	 ?the	 ?embryologic	 ?origin	 ?of	 ?these	 ?muscles,	 ?first	 ?pharyngeal	 ?arch	 ?and	 ?fourth	 ?pharyngeal	 ?arch	 ?syndromes	 ?can	 ?exhibit	 ?submucous	 ? 	 ? 13	 ?clefts	 ?of	 ?the	 ?palate	 ?and	 ?velopharyngeal	 ?insufficiency	 ?(Passos-??Bueno	 ?et	 ?al.,	 ?2009;	 ?Sadler	 ?and	 ?Langman,	 ?2010a;	 ?Sperber	 ?and	 ?Guttman,	 ?2010).	 ?	 ?1.3. Clefting of the palate 	 ?Isolated	 ?clefts	 ?of	 ?the	 ?palate	 ?are	 ?rarer	 ?than	 ?cleft	 ?lip	 ?with	 ?or	 ?without	 ?cleft	 ?palate	 ?(CL/P).	 ?CL/P	 ?occurs	 ?in	 ?approximately	 ?1:700	 ?live	 ?births	 ?(Dixon	 ?et	 ?al.,	 ?2011;	 ?Mossey	 ?et	 ?al.,	 ?2009)	 ?whereas	 ?the	 ?incidence	 ?of	 ?cleft	 ?palate	 ?only	 ?(CPO)	 ?is	 ?approximately	 ?1:1500	 ?live	 ?births	 ?(Murray	 ?and	 ?Schutte,	 ?2004).	 ?	 ?Even	 ?rarer	 ?are	 ?submucous	 ?clefts	 ?which	 ?are	 ?a	 ?microform	 ?of	 ?CPO.	 ?The	 ?incidence	 ?has	 ?been	 ?reported	 ?as	 ?anywhere	 ?from	 ?1:1250-??1:5000	 ?(Garcia	 ?Velasco	 ?et	 ?al.,	 ?1988;	 ?Weatherley-??White	 ?et	 ?al.,	 ?1972).	 ?The	 ?presentation	 ?of	 ?a	 ?submucous	 ?cleft	 ?ranges	 ?from	 ?bifid	 ?uvula	 ?which	 ?is	 ?often	 ?undiagnosed,	 ?to	 ?notching	 ?or	 ?defects	 ?of	 ?the	 ?bone	 ?of	 ?the	 ?posterior	 ?hard	 ?palate,	 ?to	 ?complete	 ?clefting	 ?of	 ?the	 ?muscles	 ?of	 ?the	 ?soft	 ?palate.	 ?	 ?Symptomatic	 ?clefts	 ?of	 ?the	 ?muscles	 ?of	 ?the	 ?soft	 ?palate	 ?are	 ?associated	 ?with	 ?velopharyngeal	 ?insufficiency	 ?(VPI),	 ?which	 ?prevents	 ?the	 ?soft	 ?palate	 ?from	 ?creating	 ?a	 ?seal	 ?between	 ?the	 ?oral	 ?and	 ?nasal	 ?cavities	 ?during	 ?function.	 ?This	 ?can	 ?cause	 ?difficulties	 ?with	 ?swallowing	 ?and	 ?hypernasal	 ?speech,	 ?and	 ?can	 ?lead	 ?to	 ?hearing	 ?loss	 ?due	 ?to	 ?chronic	 ?otitis	 ?media	 ?(Pauws	 ?et	 ?al.,	 ?2009b)	 ?.	 ?Often	 ?surgical	 ?intervention	 ?will	 ?be	 ?required	 ?to	 ?repair	 ?the	 ?defects	 ?in	 ?the	 ?muscles,	 ?however	 ?30%	 ?of	 ?all	 ?clefts	 ?with	 ?soft	 ?palate	 ?defect	 ?that	 ?are	 ?operated	 ?on	 ?will	 ?have	 ?persistent	 ?VPI	 ?and	 ?hypernasal	 ?speech	 ?(Cohen	 ?et	 ?al.,	 ?1993).	 ?	 ?	 ? 	 ? 14	 ?1.4.  Microforms of clefting  Many microforms of cleft lip have been discussed in the literature. These defects generally cause no functional impairment. Microforms of cleft lip range from notching of the upper lip, notching of the alveolar ridge +/- defects in the adjacent lateral incisor, or asymmetry in the alar cartilages (Mittal et al., 2012).  Recently, occult defects in the orbicularis oris muscle have been identified as the mildest form of cleft lip microforms.  Often children with non-syndromic cleft lip will have family members with notching of the orbiularis oris, at a rate of 13% (Mittal et al., 2012). When viewing defects of the orbicularis oris as a microform of cleft lip, it would be reasonable to consider the defects associated with submucous clefting of the palate such as bifid uvula, zona pellucida, and notching of the hard palate with absence of a posterior nasal spine as microforms of cleft palate (Ha et al., 2013). Furthermore, the occult submucous cleft palate patients who have velopharyngeal insufficiency with no identifiable anatomical defects (Kaplan, 1975) could be the most subtle microform of cleft palate.  Generally, velopharyngeal insufficiency is due to an inability of the right and left portions of the levator veli palatini muscle to contact in the midline, and thus the muscle does not elevate the palate sufficiently during function (Weatherley-White et al., 1972). The VPI microform of cleft could be due to an intrinsic defect in the levator veli palatini.  When occult submucous clefts are considered, the incidence of submucous clefting of the palate may be significantly higher than the values of 0.02-0.08% of children reported in the literature (Weatherley-White et al., 1972). Furthermore, identification of families with submucous clefts may predict individuals who are more likely to have children born with cleft palate.  	 ? 15	 ?1.5. Etiology of orofacial clefting 	 ?The	 ?etiology	 ?of	 ?clefting	 ?is	 ?multifactorial,	 ?having	 ?both	 ?genetic	 ?and	 ?environmental	 ?influences	 ?(Beaty	 ?et	 ?al.,	 ?2011;	 ?Marazita,	 ?2012),	 ?however	 ?it	 ?is	 ?thought	 ?that	 ?CPO	 ?has	 ?a	 ?larger	 ?genetic	 ?contribution.	 ?	 ?1.3-??25.3	 ?in	 ?10,000	 ?infants	 ?will	 ?be	 ?born	 ?with	 ?cleft	 ?palate	 ?only	 ?and	 ?as	 ?many	 ?as	 ?61%	 ?of	 ?cleft	 ?palate	 ?only	 ?patients	 ?have	 ?other	 ?developmental	 ?abnormalities	 ?that	 ?could	 ?be	 ?part	 ?of	 ?a	 ?syndrome	 ?(Bell	 ?et	 ?al.,	 ?2013;	 ?Mossey	 ?and	 ?Castilla,	 ?2003).	 ?Isolated	 ?cleft	 ?palate	 ?occur	 ?more	 ?frequently	 ?in	 ?females	 ?than	 ?males	 ?(Mossey	 ?and	 ?Little,	 ?2002).	 ?There	 ?are	 ?environmental	 ?factors	 ?that	 ?increase	 ?the	 ?risk	 ?of	 ?human	 ?orofacial	 ?clefting.	 ?These	 ?include	 ?teratogens	 ?as	 ?well	 ?as	 ?such	 ?factors	 ?as	 ?smoking,	 ?alcohol,	 ?maternal	 ?obesity	 ?(Dixon	 ?et	 ?al.,	 ?2011).	 ?The	 ?animal	 ?studies	 ?on	 ?teratogens	 ?causing	 ?cleft	 ?palate	 ?were	 ?recently	 ?reviewed	 ?by	 ?Barbara	 ?Abbott	 ?(Abbott,	 ?2010)	 ?.	 ?	 ?Cortisone	 ?was	 ?shown	 ?to	 ?induce	 ?cleft	 ?palates	 ?in	 ?mice	 ?in	 ?a	 ?dose-??dependent	 ?manner	 ?(Fraser	 ?and	 ?Fainstat,	 ?1951;	 ?Fraser	 ?et	 ?al.,	 ?1954).	 ?Cortisone	 ?exposure	 ?lead	 ?delayed	 ?elevation	 ?of	 ?the	 ?palatal	 ?shelves	 ?and	 ?failure	 ?to	 ?contact	 ?and	 ?fuse	 ?(Diewert	 ?and	 ?Pratt,	 ?1981;	 ?Kalter	 ?and	 ?Warkany,	 ?1961).	 ?	 ?Vitamin	 ?A	 ?(retinoic	 ?acid)	 ?exposure	 ?in	 ?rats	 ?produced	 ?a	 ?number	 ?of	 ?congenital	 ?anomalies	 ?including	 ?cleft	 ?palate	 ?(Cohlan,	 ?1953;	 ?Kalter	 ?and	 ?Warkany,	 ?1961;	 ?Walker	 ?and	 ?Crain,	 ?1960).	 ?	 ?The	 ?palatal	 ?shelves	 ?of	 ?offspring	 ?of	 ?rats	 ?exposed	 ?to	 ?vitamin	 ?A	 ?were	 ?small	 ?and	 ?abnormal	 ?in	 ?size,	 ?and	 ?failed	 ?to	 ?contact	 ?in	 ?the	 ?midline.	 ?	 ?Other	 ?teratogens	 ?cause	 ?cleft	 ?palate	 ?by	 ?impeding	 ?growth	 ?of	 ?the	 ?mandible.	 ?In	 ?such	 ?a	 ?scenario	 ?palatal	 ?shelves	 ?will	 ?remain	 ?lodged	 ?beside	 ?the	 ?tongue	 ?and	 ?will	 ?not	 ?reorient	 ?at	 ?the	 ?correct	 ?time	 ?(Diewert,	 ?1981).	 ?	 ?	 ? 	 ? 16	 ?The	 ?anti-??seizure	 ?medication	 ?Dilantin	 ?increases	 ?the	 ?risk	 ?of	 ?clefting.	 ?In	 ?a	 ?mouse	 ?study,	 ?submucous	 ?clefts	 ?were	 ?induced	 ?by	 ?exposing	 ?the	 ?pregnant	 ?mothers	 ?to	 ?high	 ?doses	 ?of	 ?oral	 ?phenytoin	 ?(Poswillo,	 ?1974).	 ?The	 ?control	 ?and	 ?experimental	 ?offspring	 ?developed	 ?in	 ?a	 ?similar	 ?fashion	 ?until	 ?the	 ?time	 ?period	 ?between	 ?days	 ?E16.5-??E18.5.	 ?At	 ?this	 ?point	 ?the	 ?ossification	 ?centers	 ?of	 ?the	 ?palatal	 ?process	 ?of	 ?the	 ?maxilla	 ?were	 ?extending	 ?medially	 ?in	 ?to	 ?each	 ?palatal	 ?shelf,	 ?however	 ?in	 ?the	 ?experimental	 ?group,	 ?the	 ?plates	 ?failed	 ?to	 ?extend	 ?all	 ?the	 ?way	 ?to	 ?the	 ?midline,	 ?and	 ?thus	 ?never	 ?contacted	 ?to	 ?create	 ?a	 ?midpalatal	 ?suture	 ?in	 ?the	 ?hard	 ?palate.	 ?	 ?Similar	 ?findings	 ?were	 ?seen	 ?in	 ?the	 ?soft	 ?palate,	 ?as	 ?there	 ?was	 ?no	 ?uvula	 ?in	 ?the	 ?experimental	 ?group	 ?and	 ?the	 ?soft	 ?palate	 ?was	 ?much	 ?thinner	 ?than	 ?the	 ?control	 ?group,	 ?representing	 ?a	 ?midline	 ?deficiency.	 ?None	 ?of	 ?the	 ?experimental	 ?groups	 ?had	 ?bifid	 ?soft	 ?palates,	 ?ie	 ?all	 ?palates	 ?fused,	 ?the	 ?clefts	 ?were	 ?only	 ?submucous.	 ?	 ?Thus	 ?in	 ?the	 ?case	 ?of	 ?seizure	 ?medications,	 ?there	 ?may	 ?be	 ?a	 ?specific	 ?risk	 ?of	 ?inducing	 ?soft	 ?palate	 ?clefting.	 ?	 ?	 ?There	 ?is	 ?also	 ?an	 ?important	 ?genetic	 ?component	 ?to	 ?the	 ?etiology	 ?of	 ?clefting.	 ?Multiple	 ?genes	 ?are	 ?likely	 ?to	 ?be	 ?involved,	 ?some	 ?of	 ?which	 ?will	 ?have	 ?mutations	 ?that	 ?lead	 ?to	 ?a	 ?change	 ?in	 ?function	 ?and	 ?others	 ?that	 ?may	 ?have	 ?subtle	 ?sequence	 ?variations	 ?that	 ?do	 ?not	 ?obviously	 ?change	 ?protein	 ?function	 ?or	 ?expression.	 ?	 ?To	 ?attack	 ?the	 ?complex	 ?nature	 ?of	 ?human	 ?clefting	 ?studies	 ?have	 ?used	 ?candidate	 ?gene	 ?approaches	 ?exploring	 ?the	 ?variation	 ?in	 ?genes	 ?known	 ?to	 ?cause	 ?human	 ?craniofacial	 ?syndromes	 ?or	 ?mouse	 ?facial	 ?phenotypes.	 ?	 ?Two	 ?syndromic	 ?genes	 ?that	 ?have	 ?been	 ?studied	 ?in	 ?isolated	 ?clefts	 ?(non-??syndromic)	 ?are	 ?the	 ?transcription	 ?factors	 ?IRF6	 ?(interferon	 ?regulatory	 ?factor	 ?6)	 ?and	 ?TBX22	 ?(T-??box	 ?transcription	 ?factor).	 ?IRF6	 ?is	 ?mutated	 ?in	 ?Van	 ?der	 ?Woude?s	 ?syndrome	 ?(Kondo	 ?et	 ?al.,	 ?2002)	 ?whereas	 ?TBX22	 ?is	 ?mutated	 ?in	 ?X-??linked	 ?cleft	 ?palate	 ?ankyloglossia	 ?(Braybrook	 ? 	 ? 17	 ?et	 ?al.,	 ?2001).	 ?	 ?In	 ?the	 ?case	 ?of	 ?IRF6,	 ?numerous	 ?studies	 ?link	 ?variation	 ?in	 ?the	 ?gene	 ?with	 ?non-??syndromic	 ?clefting	 ?(Park	 ?et	 ?al.,	 ?2007;	 ?Scapoli	 ?et	 ?al.,	 ?2005;	 ?Zucchero	 ?et	 ?al.,	 ?2004).	 ?TBX22	 ?mutations	 ?have	 ?been	 ?associated	 ?with	 ?non-??syndromic	 ?isolated	 ?cleft	 ?palate	 ?(Marcano	 ?et	 ?al.,	 ?2004;	 ?Suphapeetiporn	 ?et	 ?al.,	 ?2007).	 ?	 ?Additional	 ?genes	 ?that	 ?have	 ?been	 ?associated	 ?with	 ?non-??syndromic	 ?CPO	 ?include	 ?FOXE1,	 ?MSX1,	 ?and	 ?SATB2	 ?(Bush	 ?and	 ?Jiang,	 ?2012).	 ?	 ?These	 ?genes	 ?are	 ?involved	 ?in	 ?various	 ?stages	 ?of	 ?secondary	 ?palate	 ?development	 ?and	 ?gene	 ?targeting	 ?experiments	 ?have	 ?been	 ?shown	 ?to	 ?cause	 ?clefting	 ?in	 ?mice.	 ?	 ?	 ?More	 ?recently	 ?unbiased,	 ?genome-??wide	 ?association	 ?studies	 ?(GWAS)	 ?have	 ?been	 ?performed	 ?to	 ?identify	 ?additional	 ?variants	 ?associated	 ?with	 ?non-??syndromic	 ?orofacial	 ?clefting	 ?(Marazita,	 ?2012).	 ?	 ?To	 ?date,	 ?four	 ?studies	 ?have	 ?been	 ?performed	 ?on	 ?CL/P	 ?cases	 ?and	 ?controls	 ?(Beaty	 ?et	 ?al.,	 ?2010;	 ?Birnbaum	 ?et	 ?al.,	 ?2009;	 ?Grant	 ?et	 ?al.,	 ?2009;	 ?Mangold	 ?et	 ?al.,	 ?2010)	 ?and	 ?one	 ?on	 ?CPO	 ?cases	 ?and	 ?controls	 ?(Beaty	 ?et	 ?al.,	 ?2010;	 ?Beaty	 ?et	 ?al.,	 ?2011).	 ?	 ?The	 ?first	 ?CL/P	 ?of	 ?this	 ?kind	 ?identified	 ?IRF6	 ?as	 ?a	 ?causative	 ?gene	 ?in	 ?CL/P	 ?(Birnbaum	 ?et	 ?al.,	 ?2009).	 ?This	 ?study	 ?as	 ?well	 ?as	 ?others	 ?have	 ?linked	 ?CL/P	 ?to	 ?sequence	 ?variations	 ?on	 ?chromosome	 ?8q24	 ?which	 ?as	 ?yet	 ?is	 ?considered	 ?to	 ?be	 ?a	 ?gene	 ?desert	 ?(Beaty	 ?et	 ?al.,	 ?2010;	 ?Beaty	 ?et	 ?al.,	 ?2013).	 ?	 ?Two	 ?additional	 ?loci	 ?were	 ?found,	 ?at	 ?17q22	 ?near	 ?NOGGIN	 ?and	 ?10q25.3	 ?near	 ?VAX1	 ?(Mangold	 ?et	 ?al.,	 ?2010).	 ?	 ?One	 ?of	 ?these	 ?studies	 ?examined	 ?gene-??environment	 ?interactions	 ?and	 ?found	 ?that	 ?while	 ?no	 ?polymorphism	 ?reached	 ?significance	 ?on	 ?its	 ?own	 ?there	 ?were	 ?several	 ?that	 ?when	 ?combined	 ?with	 ?alcohol	 ?exposure	 ?or	 ?smoking	 ?increased	 ?risk	 ?of	 ?cleft	 ?palate	 ?(Beaty	 ?et	 ?al.,	 ?2011).	 ?Thus	 ?clefting	 ?remains	 ?a	 ?complicated	 ?disorder	 ?that	 ?may	 ?be	 ?difficult	 ?to	 ?tease	 ?apart.	 ?Nevertheless	 ? 	 ? 18	 ?some	 ?of	 ?the	 ?environmental	 ?risk	 ?factors	 ?could	 ?translate	 ?into	 ?recommendations	 ?for	 ?pregnant	 ?women.	 ?	 ?1.6  Aims The	 ?primary	 ?aims	 ?of	 ?my	 ?study	 ?are:	 ?	 ?1)	 ?To	 ?analyze	 ?soft	 ?palate	 ?development	 ?on	 ?a	 ?more	 ?representative	 ?group	 ?of	 ?human	 ?specimens	 ?than	 ?what	 ?was	 ?used	 ?by	 ?Burdi	 ?and	 ?Faist	 ?(Burdi	 ?and	 ?Faist,	 ?1967)	 ?2)	 ?To	 ?observe	 ?the	 ?presence	 ?or	 ?absence	 ?of	 ?midline	 ?epithelial	 ?seams	 ?between	 ?the	 ?soft	 ?palate	 ?shelves	 ?during	 ?development.	 ?	 ?3)	 ?To	 ?assess	 ?the	 ?invasion	 ?of	 ?soft	 ?palate	 ?muscles	 ?in	 ?relation	 ?to	 ?the	 ?midline	 ?seam.	 ? 	 ? 19	 ?	 ?Chapter 2 - Methods 2.1. Preparation of specimens and processing into paraffin 	 ?The	 ?specimens	 ?obtained	 ?for	 ?this	 ?study	 ?were	 ?stored	 ?in	 ?the	 ?lab	 ?of	 ?Dr.	 ?V.	 ?Diewert	 ?either	 ?as	 ?paraffin	 ?sections	 ?or	 ?as	 ?specimens	 ?stored	 ?at	 ?4?C.	 ?These	 ?non-??processed	 ?specimens	 ?were	 ?stored	 ?in	 ?4%	 ?paraformaldehyde	 ?since	 ?1988.	 ?Specimens	 ?obtained	 ?in	 ?4%	 ?PFA	 ?were	 ?washed	 ?twice	 ?in	 ?PBS	 ?for	 ?one	 ?hour	 ?each,	 ?photographed	 ?at	 ?multiple	 ?angles	 ?using	 ?a	 ?Leica	 ?stereoscope.	 ?	 ?To	 ?facilitate	 ?sectioning,	 ?it	 ?was	 ?essential	 ?to	 ?decalcify	 ?the	 ?osseous	 ?tissue.	 ?In	 ?some	 ?archival	 ?material	 ?already	 ?embedded	 ?in	 ?paraffin	 ?and	 ?sectioned	 ?there	 ?was	 ?evidence	 ?of	 ?tearing	 ?of	 ?tissues	 ?due	 ?to	 ?incomplete	 ?decalcification.	 ?	 ?	 ? All	 ?specimens	 ?were	 ?transferred	 ?into	 ?7%	 ?EDTA	 ?and	 ?placed	 ?on	 ?a	 ?shaker	 ?at	 ?4?C.	 ?This	 ?decalcification	 ?solution	 ?was	 ?changed	 ?every	 ?2-??3	 ?days	 ?for	 ?8	 ?weeks.	 ?Once	 ?it	 ?was	 ?determined	 ?that	 ?no	 ?additional	 ?bone	 ?remained	 ?in	 ?the	 ?specimens	 ?by	 ?piercing	 ?non-??critical	 ?regions	 ?of	 ?the	 ?skull	 ?with	 ?a	 ?pin,	 ?they	 ?were	 ?washed	 ?twice	 ?in	 ?PBS	 ?for	 ?one	 ?hour	 ?each,	 ?then	 ?50%PBS/50%EtOH	 ?for	 ?one	 ?hour,	 ?then	 ?70%	 ?EtOH	 ?in	 ?water.	 ?They	 ?were	 ?then	 ?processed	 ?into	 ?wax	 ?blocks	 ?at	 ?the	 ?UBC	 ?Department	 ?of	 ?Histopathology.	 ?The	 ?specimens	 ?were	 ?remelted	 ?in	 ?the	 ?lab	 ?and	 ?repositioned	 ?within	 ?the	 ?molds	 ?to	 ?ensure	 ?even	 ?transverse	 ?sections	 ?could	 ?be	 ?obtained.	 ?All	 ?specimens	 ?were	 ?sectioned	 ?at	 ?thickness	 ?of	 ?	 ?7?m	 ?on	 ?a	 ?microtome.	 ?	 ?Sections	 ?were	 ?placed	 ?on	 ?TESPA-??coated	 ?slides	 ?so	 ?that	 ?they	 ?could	 ?be	 ?used	 ?for	 ?molecular	 ?studies.	 ?	 ? 	 ? 20	 ?	 ?2.2. Histochemical and immunohistochemical staining procedures 	 ?Picrosirius	 ?red	 ?and	 ?Alcian	 ?blue	 ?staining	 ?	 ? Sections	 ?were	 ?dewaxed	 ?in	 ?two	 ?washes	 ?of	 ?xylene	 ?for	 ?20	 ?minutes	 ?each,	 ?100%	 ?EtOH	 ?for	 ?2x10	 ?minute,	 ?90%	 ?EtOH	 ?for	 ?5	 ?mintues,	 ?70%	 ?EtOH	 ?for	 ?5	 ?minutes,	 ?50%	 ?EtOH	 ?for	 ?5	 ?minutes	 ?and	 ?ddH2O	 ?for	 ?5	 ?mins.	 ?First	 ?Alcian	 ?blue	 ?staining	 ?was	 ?performed	 ?by	 ?immersing	 ?sections	 ?in	 ?	 ?1%	 ?alcian	 ?blue	 ?in	 ?acetic	 ?acid	 ?for	 ?30	 ?minutes	 ?followed	 ?by	 ?1%	 ?acetic	 ?acid	 ?for	 ?5	 ?mins,	 ?ddH2O	 ?for	 ?5	 ?mins.	 ?Next	 ?the	 ?sections	 ?were	 ?counter	 ?stained	 ?with	 ?Picrosirius	 ?red	 ?for	 ?1	 ?hour	 ?in	 ?the	 ?dark,	 ?1%	 ?acetic	 ?acid	 ?for	 ?5	 ?min,	 ?	 ?and	 ?ddH2O	 ?for	 ?5	 ?min.	 ?Specimens	 ?were	 ?rehydrated	 ?in	 ?the	 ?opposite	 ?order	 ?as	 ?they	 ?were	 ?dewaxed,	 ?then	 ?mounted	 ?on	 ?slides	 ?using	 ?Entellan	 ?mounting	 ?medium.	 ?	 ?Hematoxylin	 ?and	 ?Eosin	 ?staining	 ?	 ? The	 ?dewaxing	 ?protocol	 ?was	 ?performed	 ?in	 ?the	 ?same	 ?manner	 ?as	 ?Picrosirius	 ?red	 ?and	 ?Alcian	 ?blue	 ?staining.	 ?Slides	 ?were	 ?then	 ?stained	 ?in	 ?Shandon	 ?hematoxylin	 ?for	 ?5-??7	 ?minutes,	 ?ddH2O	 ?for	 ?5	 ?minutes,	 ?immersed	 ?in	 ?in saturated lithium carbonate solution for 1 minute to blue the hematoxylin, dipped in 1%	 ?Eosin	 ?Y	 ?aqueous	 ?solution	 ?30	 ?times	 ?and	 ?rinsed	 ?in	 ?ddH2O	 ?for	 ?5	 ?minutes.	 ?Specimens	 ?were	 ?dehydrated	 ?in	 ?the	 ?opposite	 ?order	 ?as	 ?they	 ?were	 ?dewaxed	 ?and	 ?coverslipped	 ?as	 ?described	 ?above.	 ?	 ?	 ? 	 ? 21	 ?Immunohistochemistry	 ?protocol	 ?for	 ?MF20	 ?muscle	 ?antibody	 ?The	 ?MF20	 ?mouse	 ?monoclonal	 ?antibody	 ?was	 ?obtained	 ?from	 ?the	 ?Developmental	 ?Studies	 ?Hybridoma	 ?bank.	 ?This	 ?antibody	 ?is	 ?raised	 ?to	 ?chicken	 ?myosin	 ?and	 ?cross	 ?reacts	 ?with	 ?a	 ?wide	 ?variety	 ?of	 ?species.	 ?Sections	 ?were	 ?dewaxed,	 ?rehydrated	 ?to	 ?50%	 ?ethanol	 ?and	 ?rinsed	 ?in	 ?PBS	 ?three	 ?times	 ?for	 ?2	 ?minutes	 ?each.	 ?A	 ?Proteinase	 ?K	 ?treatment	 ?was	 ?performed	 ?for	 ?10	 ?minutes	 ?at	 ?37?	 ?C	 ?using	 ?5?g/ml	 ?in	 ?Proteinase	 ?K	 ?buffer.	 ?Sections	 ?were	 ?rinsed	 ?again	 ?in	 ?PBS	 ?three	 ?times.	 ?Blocking	 ?was	 ?performed	 ?with	 ?1%BSA,	 ?0.02%	 ?TWEEN	 ?and	 ?1%	 ?sheep	 ?serum	 ?for	 ?30	 ?minutes	 ?at	 ?room	 ?temperature	 ?in	 ?a	 ?humidified	 ?chamber.	 ?The	 ?MF	 ?20	 ?primary	 ?antibody	 ?(supernatant)	 ?was	 ?added	 ?undiluted	 ?to	 ?the	 ?sections	 ?and	 ?left	 ?for	 ?one	 ?hour	 ?at	 ?37?C.	 ?It	 ?was	 ?then	 ?rinsed	 ?in	 ?PBS	 ?again	 ?three	 ?times.	 ?	 ?The	 ?secondary	 ?antibody	 ?used	 ?was	 ?anti-??mouse	 ?IgG	 ?biotinylated	 ?antibody	 ?1:500	 ?in	 ?PBS	 ?and	 ?0.2%	 ?BSA	 ?(ABC	 ?kit,	 ?Vector	 ?Labs)	 ?and	 ?was	 ?added	 ?to	 ?the	 ?sections	 ?for	 ?one	 ?hour	 ?at	 ?room	 ?temperature.	 ?Again	 ?sections	 ?were	 ?rinsed	 ?three	 ?times	 ?in	 ?PBS.	 ?From	 ?the	 ?ABC	 ?Kit,	 ?1	 ?drop	 ?of	 ?A	 ?and	 ?1	 ?drop	 ?of	 ?B	 ?were	 ?added	 ?to	 ?3.2mL	 ?PBS	 ?30	 ?minutes	 ?before	 ?needed	 ?for	 ?use,	 ?then	 ?incubated	 ?with	 ?the	 ?sections	 ?for	 ?one	 ?hour	 ?at	 ?room	 ?temperature.	 ?DAB	 ?detection	 ?was	 ?performed	 ?in	 ?the	 ?dark	 ?at	 ?room	 ?temperature.	 ?The	 ?detection	 ?solution	 ?(200?L	 ?50x	 ?DAB	 ?and	 ?10mL	 ?1x	 ?DAB	 ?buffer)	 ?was	 ?applied	 ?to	 ?slides	 ?and	 ?reaction	 ?was	 ?monitored.	 ?The	 ?detection	 ?solution	 ?was	 ?left	 ?on	 ?slides	 ?for	 ?10-??20	 ?minutes	 ?until	 ?a	 ?full	 ?reaction	 ?was	 ?observed.	 ?The	 ?reaction	 ?was	 ?stopped	 ?by	 ?washing	 ?in	 ?water	 ?three	 ?times	 ?for	 ?one	 ?minute	 ?each,	 ?then	 ?washed	 ?in	 ?distilled	 ?water	 ?for	 ?5	 ?minutes.	 ?Conterstaining	 ?was	 ?done	 ?with	 ?Picrosirius	 ?red	 ?and	 ?Alcian	 ?blue	 ?and	 ?slides	 ?were	 ?prepared	 ?as	 ?per	 ?above	 ?protocol.	 ?	 ?	 ? 	 ? 22	 ?2.3. 3D reconstruction of human fetuses at various stages of palatogenesis 	 ?Thirteen	 ?sectioned	 ?and	 ?stained	 ?human	 ?specimens	 ?at	 ?54-??74-??days	 ?of	 ?development	 ?were	 ?photographed	 ?serially	 ?at	 ?the	 ?hard-??soft	 ?palate	 ?junction	 ?on	 ?a	 ?Zeiss	 ?Axiophot	 ?compound	 ?microscope.	 ?These	 ?high	 ?resolution	 ?images	 ?were	 ?imported	 ?in	 ?to	 ?WinSurf	 ?3D	 ?Reconstruction	 ?program	 ?(SURFdriver	 ?Software,	 ?Kailua,	 ?HI,	 ?USA	 ?developed	 ?by	 ?Scott	 ?Lozanoff).	 ?The	 ?structures	 ?of	 ?interest	 ?included	 ?the	 ?hard	 ?palate,	 ?soft	 ?palate,	 ?palatine	 ?bones,	 ?midline	 ?epithelial	 ?seam,	 ?palatine	 ?aponeurosis,	 ?and	 ?the	 ?nasal	 ?septum.	 ?	 ?These	 ?structures	 ?were	 ?traced	 ?on	 ?the	 ?sequential	 ?photos	 ?for	 ?each	 ?specimen.	 ?	 ?The	 ?tracings	 ?were	 ?then	 ?stacked	 ?together	 ?to	 ?create	 ?the	 ?reconstructed	 ?structures.	 ?The	 ?stacks	 ?were	 ?aligned	 ?within	 ?the	 ?Winsurf	 ?program	 ?and	 ?smoothed	 ?to	 ?create	 ?a	 ?representation	 ?of	 ?the	 ?hard-??soft	 ?palate	 ?junction.	 ?Screen	 ?captures	 ?were	 ?created	 ?from	 ?different	 ?views	 ?of	 ?the	 ?reconstruction	 ?and	 ?imported	 ?into	 ?Adobe	 ?Photoshop.	 ? 	 ? 23	 ?	 ?Chapter 3: Results  3.1. Overview of craniofacial development from 54 ? 74-days post conception 	 ?In	 ?order	 ?to	 ?gain	 ?an	 ?overview	 ?of	 ?the	 ?morphogenesis	 ?of	 ?the	 ?human	 ?head	 ?I	 ?will	 ?describe	 ?the	 ?major	 ?structures	 ?that	 ?are	 ?developing	 ?at	 ?each	 ?stage	 ?of	 ?development	 ?encompassed	 ?in	 ?my	 ?sample.	 ?The	 ?bones,	 ?cartilages,	 ?teeth,	 ?muscles	 ?and	 ?salivary	 ?glands	 ?are	 ?all	 ?present	 ?in	 ?the	 ?specimens	 ?and	 ?help	 ?to	 ?provide	 ?another	 ?measure	 ?of	 ?the	 ?state	 ?of	 ?development	 ?in	 ?addition	 ?to	 ?the	 ?stage	 ?of	 ?palate	 ?closure.	 ?The	 ?specimens	 ?collected	 ?had	 ?crown	 ?rump	 ?lengths	 ?ranging	 ?from	 ?35	 ?to	 ?75	 ?mm	 ?(data	 ?obtained	 ?from	 ?U	 ?of	 ?Washington,	 ?Table	 ?3.1,	 ?3.3).	 ?The	 ?stages	 ?I	 ?examined	 ?cover	 ?those	 ?included	 ?in	 ?the	 ?Burdi	 ?study	 ?but	 ?expand	 ?the	 ?numbers	 ?in	 ?41-??70	 ?mm	 ?CR	 ?length	 ?which	 ?is	 ?the	 ?critical	 ?period	 ?covering	 ?soft	 ?palate	 ?fusion	 ?(Table	 ?3.2).	 ?Sex	 ?information	 ?for	 ?the	 ?individual	 ?specimens	 ?was	 ?not	 ?available.	 ?	 ? 	 ? 24	 ?	 ? 	 ?Table	 ?3.1	 ?Palate	 ?development	 ?in	 ?individual	 ?human	 ?embryos	 ?and	 ?fetuses	 ?Specimen number Days post conception Crown-rump length (mm) as per UW Hard palate (fused with seam, seam has epithelial remnants, no seam fully fused) Soft palate (not fused, partially fused with seam, fused with seam, fused with aponeurosis)    Posterior Anterior Middle posterior H7832 54d 35 Fused with seam Fused with seam Partially fused with seam Not fused H9978 54d 35 Fused with seam No sections in soft palate, excluded from sample H9517 57d 41 Fused with seam Partially fused with seam Partially fused with seam Not fused H10142 57d 41 Fused with remnants of seam Fused with remnants of seam Fused with remnants of seam Not fused H10458 57d 41 Fused with seam Fused with remnants of seam Fused with remnants of seam Not fused H11032 57d 41 Fused with seam Fused with remnants of seam Partially fused with seam Not fused H10593 59d 42 Fused with seam Not fused Not fused Not fused H9652 59d 42 Fused no seam Fused no seam, developing aponeurosis Fused no seam, developing aponeurosis N/A H9854 64d 43-62 Fused with remnants of seam Fused no seam, developing aponeurosis Partially fused with seam Not fused H10071 67d 63 Fused with remnants of seam Fused no seam, developing aponeurosis Fused no seam, developing aponeurosis Not fused H9705 70d 64-68 Fused with remnants of seam Fused no seam, developing aponeurosis Fused no seam, developing aponeurosis Fused with seam H9832 70d 64-68 Fused with remnants of seam Fused with aponeurosis N/A N/A H9819 74d 75 Epithelial pearls Fused with aponeurosis Fused with aponeurosis Fused with aponeurosis  	 ? 25	 ? 	 ?Table	 ?3.2	 ?Comparison	 ?of	 ?crown-?rump	 ?length	 ?of	 ?specimens	 ?from	 ?current	 ?study	 ?with	 ?Burdi	 ?and	 ?Faist	 ?1967	 ?study	 ?Crown-rump length (mm) My study (n=) Burdi and Faist (n=) 35-40 2 7 41-60* 6 2 61-70* 4 0 71-75 1 1  * Key stages of soft palate seam formation 	 ? 	 ? 26	 ?	 ? 3.1.1 Tooth development 	 ?	 ? The	 ?stages	 ?of	 ?individual	 ?tooth	 ?development	 ?pass	 ?through	 ?initiation,	 ?morphogenesis,	 ?histodifferentiation	 ?and	 ?eruption.	 ?The	 ?enamel	 ?of	 ?teeth	 ?is	 ?formed	 ?from	 ?ectoderm,	 ?and	 ?the	 ?dentin	 ?and	 ?pulp	 ?are	 ?made	 ?from	 ?ectomesenchyme	 ?of	 ?neural	 ?crest	 ?origin.	 ?The	 ?communication	 ?between	 ?these	 ?two	 ?layers	 ?is	 ?critical	 ?for	 ?regulating	 ?the	 ?signaling	 ?mechanisms	 ?involved	 ?in	 ?tooth	 ?development	 ?(Jussila	 ?and	 ?Thesleff,	 ?2012).	 ?Tooth	 ?development	 ?with	 ?dental	 ?lamina	 ?formation	 ?early	 ?as	 ?28	 ?days	 ?pc,	 ?and	 ?root	 ?development	 ?continues	 ?until	 ?3	 ?years	 ?post-??eruption	 ?(Oo?,	 ?1981).	 ?Initially,	 ?teeth	 ?begin	 ?as	 ?dental	 ?placodes,	 ?which	 ?are	 ?localized	 ?epithelial	 ?thickenings	 ?within	 ?the	 ?dental	 ?lamina.	 ?In	 ?the	 ?specimens	 ?studied	 ?here	 ?teeth	 ?reach	 ?bud,	 ?cap	 ?and	 ?bell	 ?stage.	 ?In	 ?the	 ?54	 ?and	 ?57-??day	 ?specimens	 ?there	 ?are	 ?some	 ?bud	 ?and	 ?cap	 ?stage	 ?teeth	 ?(Fig.	 ?3.1A,B).	 ?In	 ?the	 ?64,	 ?67	 ?and	 ?70-??day	 ?specimens	 ?there	 ?are	 ?teeth	 ?at	 ?bud,	 ?cap	 ?and	 ?early	 ?bell	 ?stage	 ?(Fig.	 ?3.	 ?1C,D,E,E?).	 ?At	 ?no	 ?time	 ?was	 ?histodifferentiation	 ?observed.	 ?	 ?	 ?	 ?3.1.2 Cartilages  	 ?	 ? The	 ?vertebrate	 ?skull	 ?is	 ?divided	 ?into	 ?three	 ?main	 ?parts:	 ?the	 ?splanchnocranium	 ?visceral	 ?skeleton),	 ?the	 ?dermatocranium	 ?(membranous	 ?bones)	 ?and	 ?the	 ?chondrocranium.	 ?The	 ?chondrocranium,	 ?or	 ?neurocranium,	 ?is	 ?the	 ?primitive	 ?skull	 ?that	 ?is	 ?evolutionarily	 ?conserved	 ?amongst	 ?all	 ?vertebrates	 ?(Sperber	 ?and	 ?Guttman,	 ?2010).	 ?	 ? 	 ? 27	 ?The	 ?function	 ?of	 ?the	 ?chondrocranium	 ?is	 ?to	 ?support	 ?the	 ?brain	 ?and	 ?sensory	 ?organs	 ?of	 ?the	 ?skull.	 ?In	 ?cartilaginous	 ?fishes,	 ?the	 ?chondrocranium	 ?will	 ?persist	 ?as	 ?cartilage,	 ?however	 ?in	 ?bony	 ?fishes	 ?and	 ?higher	 ?vertebrates	 ?the	 ?cartilage	 ?of	 ?neural	 ?crest	 ?origin	 ?will	 ?ossify	 ?by	 ?endochondral	 ?ossification.	 ?The	 ?bones	 ?of	 ?the	 ?chondrocranium	 ?in	 ?humans	 ?make	 ?up	 ?the	 ?cranial	 ?base	 ?(Sadler	 ?and	 ?Langman,	 ?2010b).	 ?In	 ?the	 ?present	 ?study	 ?it	 ?is	 ?possible	 ?the	 ?entirety	 ?of	 ?the	 ?nasal	 ?capsule	 ?including	 ?turbinates,	 ?septum	 ?and	 ?ethmoid	 ?(Fig.	 ?3.1A-??E).	 ?The	 ?orbital	 ?cartilage	 ?and	 ?Meckel?s	 ?cartilage	 ?are	 ?also	 ?differentiated	 ?	 ?(Fig.	 ?3.1A-??E).	 ?	 ?3.1.3 Bone development 	 ?	 ? The	 ?majority	 ?of	 ?the	 ?bones	 ?of	 ?the	 ?face	 ?and	 ?cranial	 ?vault	 ?develop	 ?by	 ?purely	 ?intramembranous	 ?formation	 ?(frontal,	 ?parietal,	 ?nasal,	 ?lacrimal,	 ?zygomatic,	 ?vomer,	 ?palatine	 ?and	 ?maxilla)(Sperber	 ?and	 ?Guttman,	 ?2010).	 ?Others	 ?develop	 ?through	 ?purely	 ?endochondral	 ?formation	 ?(incus,	 ?stapes,	 ?ethmoid	 ?inferior	 ?concha	 ?and	 ?hyoid).	 ?The	 ?remaining	 ?craniofacial	 ?bones	 ?develop	 ?by	 ?a	 ?mixed	 ?endochondral-??intramembranous	 ?combination	 ?(maleus,	 ?temporal,	 ?sphenoid,	 ?mandible	 ?and	 ?occipital).	 ?	 ?	 ?	 ?Even	 ?in	 ?the	 ?youngest	 ?54-??day	 ?specimen,	 ?there	 ?is	 ?a	 ?significant	 ?amount	 ?of	 ?ossification.	 ?The	 ?palatine	 ?processes	 ?of	 ?the	 ?maxilla	 ?are	 ?ossifying.	 ?Ossification	 ?of	 ?the	 ?mandibular	 ?bone	 ?is	 ?occurring	 ?around	 ?Meckel?s	 ?cartilage.	 ?The	 ?transverse	 ?portion	 ?of	 ?the	 ?frontal	 ?bone	 ?making	 ?up	 ?the	 ?superior	 ?orbital	 ?wall	 ?has	 ?also	 ?undergone	 ?significant	 ? 	 ? 28	 ?ossification	 ?and	 ?the	 ?paired	 ?ossification	 ?centers	 ?of	 ?the	 ?vomer	 ?are	 ?present.	 ?By	 ?70-??days	 ?the	 ?majority	 ?of	 ?the	 ?facial	 ?bones	 ?are	 ?present	 ?(Fig.	 ?3.1A-??E).	 ?	 ?3.1.4  Muscle development 	 ?	 ? There	 ?are	 ?approximately	 ?60	 ?muscles	 ?in	 ?the	 ?human	 ?head,	 ?which	 ?are	 ?crucial	 ?to	 ?our	 ?ability	 ?to	 ?move	 ?the	 ?head,	 ?swallow,	 ?and	 ?move	 ?our	 ?eyes.	 ?The	 ?voluntary	 ?muscles	 ?of	 ?the	 ?head	 ?and	 ?neck	 ?develop	 ?from	 ?the	 ?somitomeres	 ?and	 ?somites	 ?which	 ?are	 ?composed	 ?of	 ?paraxial	 ?mesoderm	 ?(Sperber	 ?and	 ?Guttman,	 ?2010).	 ?The	 ?extraocular	 ?muscles	 ?are	 ?the	 ?first	 ?to	 ?form	 ?and	 ?begin	 ?to	 ?develop	 ?at	 ?3-??4	 ?weeks	 ?post	 ?conception	 ?(Sevel,	 ?1981).	 ?	 ?	 ?Extraocular	 ?muscles	 ?arise	 ?from	 ?mesoderm	 ?in	 ?three	 ?different	 ?centers	 ?of	 ?development,	 ?based	 ?on	 ?the	 ?cranial	 ?nerves	 ?that	 ?innervate	 ?them:	 ?one	 ?for	 ?the	 ?muscles	 ?that	 ?are	 ?innervated	 ?by	 ?cranial	 ?nerve	 ?III	 ?(oculomotor):	 ?superior,	 ?medial	 ?and	 ?inferior	 ?rectus	 ?and	 ?the	 ?inferior	 ?oblique,	 ?and	 ?separate	 ?areas	 ?for	 ?the	 ?superior	 ?oblique	 ?(CN	 ?IV)	 ?and	 ?lateral	 ?rectus	 ?(CN	 ?VI)	 ?(Gilbert,	 ?1952).	 ?The	 ?medial	 ?rectus,	 ?superior	 ?oblique,	 ?inferior	 ?rectus	 ?are	 ?present	 ?in	 ?54-??day	 ?specimens	 ?(Fig.	 ?3.1A).	 ?	 ?The	 ?other	 ?muscles	 ?that	 ?are	 ?present	 ?at	 ?54-??days	 ?include	 ?the	 ?masseter,	 ?tongue,	 ?myelohyoid	 ?and	 ?anterior	 ?belly	 ?of	 ?the	 ?digastric	 ?(Fig.	 ?3.1A).	 ?Some	 ?of	 ?these	 ?muscles	 ?were	 ?already	 ?forming	 ?in	 ?the	 ?head	 ?during	 ?the	 ?embryonic	 ?period.	 ?	 ?For	 ?example,	 ?the	 ?muscles	 ?of	 ?the	 ?tongue	 ?(except	 ?for	 ?the	 ?palatoglossus)	 ?migrate	 ?into	 ?the	 ?tongue	 ?swellings	 ?beginning	 ?at	 ?5	 ?weeks	 ?	 ?(Sperber	 ?and	 ?Guttman,	 ?2010).	 ?	 ?The	 ?origins	 ?of	 ?the	 ?tongue	 ?musculature	 ?are	 ?the	 ?pharynx	 ?at	 ?the	 ?level	 ?of	 ?the	 ?occipital	 ?somites.	 ?	 ? 	 ? 29	 ?The	 ?muscles	 ?of	 ?mastication	 ?include	 ?the	 ?temporalis,	 ?masseter,	 ?pterygoids,	 ?anterior	 ?belly	 ?of	 ?the	 ?digastric,	 ?mylohyoid,	 ?tensor	 ?veli	 ?palatini	 ?and	 ?tensor	 ?tympani.	 ?With	 ?the	 ?exception	 ?of	 ?the	 ?TVP,	 ?muscles	 ?of	 ?mastication	 ?develop	 ?before	 ?the	 ?muscles	 ?of	 ?the	 ?soft	 ?palate	 ?(Sperber	 ?and	 ?Guttman,	 ?2010),	 ?but	 ?after	 ?the	 ?development	 ?of	 ?the	 ?mandible	 ?has	 ?begun	 ?(Cohen	 ?et	 ?al.,	 ?1993).	 ?Their	 ?origin	 ?is	 ?the	 ?first	 ?pharyngeal	 ?arch,	 ?thus	 ?they	 ?receive	 ?innervation	 ?from	 ?the	 ?mandibular	 ?branch	 ?of	 ?the	 ?trigeminal	 ?nerve	 ?(Sadler	 ?and	 ?Langman,	 ?2010b)	 ?The	 ?tongue	 ?is	 ?also	 ?prominent	 ?in	 ?all	 ?stages	 ?examined	 ?in	 ?my	 ?study.	 ?This	 ?is	 ?not	 ?surprising	 ?since	 ?the	 ?tongue	 ?initiates	 ?during	 ?the	 ?fourth	 ?weeks	 ?as	 ?lingual	 ?swellings	 ?from	 ?the	 ?first	 ?arch	 ?(Sperber	 ?and	 ?Guttman,	 ?2010).	 ?	 ?The	 ?muscles	 ?of	 ?the	 ?soft	 ?palate	 ?originate	 ?from	 ?mesenchymal	 ?tissue	 ?of	 ?the	 ?first	 ?and	 ?fourth	 ?arches	 ?(Sperber	 ?and	 ?Guttman,	 ?2010).	 ?By	 ?16-??17	 ?weeks,	 ?the	 ?full	 ?postnatal	 ?soft	 ?palate	 ?musculature	 ?is	 ?developed	 ?(Cohen	 ?et	 ?al.,	 ?1993).	 ?The	 ?first	 ?soft	 ?palate	 ?muscle	 ?to	 ?invade	 ?the	 ?palatal	 ?shelf	 ?mesenchyme	 ?is	 ?the	 ?tensor	 ?veli	 ?palatini	 ?at	 ?40	 ?days	 ?pc.	 ?Next	 ?is	 ?the	 ?palatopharyngeus	 ?at	 ?45	 ?days,	 ?the	 ?levator	 ?veli	 ?palatini	 ?during	 ?the	 ?8th	 ?week,	 ?while	 ?the	 ?palatoglossus	 ?and	 ?the	 ?musculus	 ?uvulus	 ?are	 ?the	 ?last	 ?to	 ?form	 ?during	 ?the	 ?9th	 ?week	 ?(Cohen	 ?et	 ?al.,	 ?1993;	 ?De	 ?la	 ?Cuadra	 ?Blanco	 ?et	 ?al.,	 ?2012;	 ?Katori	 ?et	 ?al.,	 ?2011;	 ?Rood,	 ?1973;	 ?Seif	 ?and	 ?Dellon,	 ?1978;	 ?Sperber	 ?and	 ?Guttman,	 ?2010).	 ?The	 ?palatoglossus	 ?does	 ?not	 ?attach	 ?to	 ?the	 ?soft	 ?palate	 ?until	 ?the	 ?11th	 ?week,	 ?as	 ?it	 ?originally	 ?develops	 ?with	 ?the	 ?muscles	 ?of	 ?the	 ?tongue	 ?(Sperber	 ?and	 ?Guttman,	 ?2010).	 ?Therefore	 ?all	 ?of	 ?these	 ?muscles	 ?are	 ?forming	 ?in	 ?our	 ?54-??day	 ?specimen	 ?(Fig.	 ?3.1A	 ?and	 ?data	 ?not	 ?shown).	 ?The	 ?tensor	 ?veli	 ?palatini	 ?muscle	 ?migrates	 ?to	 ?the	 ?precursor	 ?to	 ?the	 ?palatine	 ?aponeurosis	 ?during	 ?the	 ?8th	 ?week.	 ?By	 ?the	 ?9th	 ?week,	 ?the	 ?tensor	 ?veli	 ?palatini	 ?is	 ?continuous	 ?with	 ?the	 ? 	 ? 30	 ?aponeurosis	 ?(De	 ?la	 ?Cuadra	 ?Blanco	 ?et	 ?al.,	 ?2012).	 ?More	 ?focused	 ?descriptions	 ?of	 ?the	 ?soft	 ?palate	 ?including	 ?seam	 ?formation,	 ?invasion	 ?of	 ?soft	 ?palate	 ?muscles	 ?and	 ?insertion	 ?of	 ?muscles	 ?into	 ?the	 ?aponeurosis	 ?follows	 ?in	 ?subsequent	 ?sections	 ?of	 ?the	 ?results	 ?(Figs.	 ?3.2-??3.5,3.7).	 ?I	 ?conclude	 ?that	 ?the	 ?specimens	 ?I	 ?am	 ?studying	 ?are	 ?grossly	 ?consistent	 ?with	 ?data	 ?from	 ?others	 ?for	 ?the	 ?equivalent	 ?stages	 ?of	 ?development.	 ?	 ? 	 ? 31	 ?	 ?Figure	 ?3.1.	 ?Overview	 ?of	 ?craniofacial	 ?development	 ?in	 ?the	 ?conceptuses	 ?used	 ?in	 ?this	 ?study.	 ? 	 ?	 ? 	 ?Frontal sections of human fetuses at different stages of development, sectioned through the posterior hard palate.  A) A specimen stained with Picrosirius Red and Alcian Blue. The shelves of the hard palate are fusing in the midline and the presence of a continuous midline epithelial seam is observed (arrows). The hard palate has already fused with the nasal septum.  Cartilages and intramembranous bones have differentiated in the upper and lower jaws. The extraocular and tongue muscles are present. A maxillary right primary molar is seen in the bell stage of development while the other three primary molars are in cap stage. B) A specimen stained with hematoxylin and eosin. The palatal shelves have contacted in the midline and a continuous seam is present between the palatal shelves (arrows). The attachment of the shelves to the nasal septum appears tenuous with a continuous epithelial layer separating the structures. Similar cartilage, bone and muscle development is seen as in the 54-day specimen, however the masseter muscles can now be. Two bud stage molars can be seen developing in the right and left maxillae. C) A specimen stained with Picrosirius Red and Alcian Blue. The seam in the hard palate is beginning to break down and shows collections of epithelial cells (black arrowheads) There is increased ossification compared to younger specimens. D) A specimen stained with Picrosirius Red and Alcian Blue. The medial edge epithelium between the palatal shelves is degrading, with islands remaining at the superior and inferior aspects of the fusing shelves. The palatine process of the maxillary bone is infiltrating the hard palate.  Cap stage teeth are seen on both sides of the maxilla. E) A specimen stained with hematoxylin and eosin which was cut at an angle.  The medial edge epithelium is almost completely degraded, with a few epithelial remnants persisting in the oral side of the midline seam of the hard palate (arrows). A primary tooth in bell stage is seen in the upper right maxilla (black box). F. Area inside the black box of E. This enamel organ and dental papilla of the primary molar are clearly visible but no dentin or enamel has been deposited. The successional lamina of the developing premolar is also visible lateral to the developing primary molar. Key: bell ? bell stage tooth bud, cap ? cap stage tooth bud, dp ? dental papilla,  iee ? inner enamel epithelium, ir - inferior rectus, mes ? midline epithelial seam, mp - medial pterygoid, mr ? medial rectus, oee ? outer enamel epithelium, ppm ? palatine process of the maxilla, so ? superior oblique, sr ? stellate reticulum, sre ? superior rectus, t ? tongue, v ? vomer. Scale bar =1mm for panels A-E and 200 ?m.	 ? 	 ? 32	 ?3.2. Palate development according to stage of embryo 3.2.1 54-day specimens have incomplete fusion of the hard and soft palates 	 ?The	 ?embryonic	 ?period	 ?is	 ?nearly	 ?complete	 ?by	 ?54-??days	 ?gestation.	 ?The	 ?primary	 ?palate	 ?has	 ?fully	 ?fused	 ?by	 ?47	 ?days	 ?(Oo?,	 ?1981),	 ?	 ?the	 ?palatal	 ?shelves	 ?have	 ?reoriented	 ?across	 ?the	 ?oral	 ?cavity	 ?and	 ?are	 ?undergoing	 ?fusion	 ?with	 ?the	 ?primary	 ?palate.	 ?The	 ?shelves	 ?have	 ?contacted	 ?and	 ?largely	 ?completed	 ?fusion	 ?in	 ?the	 ?hard	 ?palate,	 ?however	 ?the	 ?epithelial	 ?seam	 ?may	 ?still	 ?be	 ?present.	 ?Two	 ?54-??day	 ?specimens	 ?were	 ?examined	 ?(Table	 ?3.1,	 ?3.3).	 ?The	 ?region	 ?from	 ?the	 ?posterior	 ?hard	 ?palate	 ?to	 ?the	 ?termination	 ?of	 ?the	 ?soft	 ?palate	 ?were	 ?examined.	 ?The	 ?Atlas	 ?of	 ?Developmental	 ?Anatomy	 ?of	 ?the	 ?Face	 ?(Kraus	 ?et	 ?al.,	 ?1966)	 ?was	 ?used	 ?to	 ?accurately	 ?determine	 ?when	 ?the	 ?transition	 ?from	 ?soft	 ?to	 ?hard	 ?palate	 ?occurred.	 ?The	 ?eyes,	 ?optic	 ?nerves	 ?and	 ?nasal	 ?septum	 ?were	 ?used	 ?to	 ?determine	 ?the	 ?depth	 ?of	 ?the	 ?sections.	 ?Other	 ?landmarks	 ?included	 ?the	 ?transition	 ?from	 ?a	 ?vertical	 ?orientation	 ?of	 ?the	 ?palatal	 ?nerve	 ?to	 ?a	 ?horizontal	 ?position	 ?as	 ?it	 ?enters	 ?the	 ?soft	 ?palate,	 ?and	 ?the	 ?presence	 ?or	 ?absence	 ?of	 ?	 ?horizontal	 ?palatine	 ?bones	 ?in	 ?the	 ?palatine	 ?shelves.	 ?	 ?One	 ?specimen	 ?(Figure	 ?3.1)	 ?was	 ?intact	 ?but	 ?the	 ?other	 ?was	 ?lacking	 ?the	 ?majority	 ?of	 ?the	 ?soft	 ?palate	 ?(data	 ?not	 ?shown,	 ?H9978).	 ?In	 ?the	 ?intact	 ?specimen	 ?a	 ?midline	 ?seam	 ?is	 ?present	 ?in	 ?the	 ?hard	 ?palate	 ?but	 ?is	 ?mostly	 ?degraded	 ?with	 ?thickened	 ?epithelium	 ?on	 ?the	 ?superior	 ?and	 ?inferior	 ?aspects	 ?(Fig.	 ?3.1A,A?).	 ?Some	 ?epithelial	 ?remnants	 ?are	 ?present	 ?in	 ?the	 ?centre	 ?of	 ?the	 ?hard	 ?palate	 ?(Fig.	 ?3.1A?).	 ?In	 ?the	 ?soft	 ?palate,	 ?the	 ?shelves	 ?have	 ?made	 ?contact	 ?and	 ?an	 ?epithelial	 ?seam	 ?runs	 ?through	 ?the	 ?midline	 ?(Fig.	 ?3.1B,B?).	 ?The	 ?same	 ?thickened	 ?areas	 ?of	 ?epithelium	 ?exist	 ?on	 ?the	 ?nasal	 ?and	 ?oral	 ?aspects.	 ?Mesenchymal	 ?condensations	 ?lateral	 ?to	 ?the	 ?midline	 ?represent	 ?the	 ?developing	 ?palatine	 ?aponeurosis.	 ? 	 ? 33	 ?The	 ?posterior	 ?soft	 ?palate	 ?(C)	 ?exhibits	 ?a	 ?lobular	 ?shape,	 ?most	 ?likely	 ?due	 ?to	 ?the	 ?curve	 ?of	 ?the	 ?soft	 ?palate	 ?as	 ?it	 ?projects	 ?down	 ?towards	 ?the	 ?uvula.	 ?The	 ?midline	 ?epithelium	 ?is	 ?thicker	 ?and	 ?more	 ?irregular	 ?in	 ?this	 ?region.	 ?It	 ?is	 ?not	 ?possible	 ?to	 ?see	 ?the	 ?developing	 ?aponeurosis	 ?in	 ?this	 ?region,	 ?but	 ?distinct	 ?lateral	 ?condensations	 ?in	 ?the	 ?soft	 ?palate	 ?shelves	 ?represent	 ?the	 ?developing	 ?tensor	 ?veli	 ?palatine	 ?muscles.	 ?It	 ?is	 ?not	 ?possible	 ?conclude	 ?based	 ?on	 ?the	 ?single	 ?specimen	 ?whether	 ?the	 ?extent	 ?of	 ?fusion	 ?of	 ?the	 ?soft	 ?palate	 ?is	 ?representative	 ?for	 ?this	 ?stage	 ? 	 ? 34	 ?	 ? 	 ?Table	 ?3.3	 ?Fusion	 ?status	 ?of	 ?the	 ?hard	 ?and	 ?soft	 ?palate	 ?in	 ?University	 ?of	 ?Washington	 ?specimens	 ?Hard Palate  Seam Epithelial remnants No seam remaining 54d (n = 2) 2 0 0 57d (n=4) 3 1 0 59d (n=2) 1 0 1 64d (n=1) 1 0 0 67d (n=1) 0 1 0 70d (n=2) 0 2 0 74d (n=1) 0 0 1 Soft Palate Days post conception Not fused Partially fused with seam Fused with seam Fused with aponeurosis 54d  (n=1) 0 1 0 0 57d (n=4) 0 1 3 0 59d (n=2) 1 0 0 1 64d (n=1) 0 1 0 0 67d (n=1) 0 0 0 1 70d (n=2) 0 0 1 1 74d (n=1) 0 0 0 1 	 ? 	 ? 35	 ?Figure	 ?3.2.	 ?Palate	 ?morphogenesis	 ?in	 ?a	 ?54-?day	 ?specimen.	 ?	 ?Frontal sections of a 54-day specimen beginning in the posterior hard palate and ending at the posterior soft palate stained with Picrosirius Red and Alcian Blue, Overall this specimen is precocious in its palate development. At the posterior edge of the hard palate (A, A?), the midline seam is still intact on the inferior and superior aspects, but is degrading in the centre (arrows). In the anterior soft palate (B, B?), the midline palatal seam is visible on the oral and nasal surfaces aspects. The intact regions of the seam are much bulkier than in the hard palate. Epithelial islands (white arrowhead) are present in the middle of the seam. The palatine aponeurosis is seen condensing within the right and left palatal shelves (black arrowhead). In the posterior soft palate (C, C?), the midline palatal seam is thicker than in more anterior regions. The epithelium is multilayered and poorly organized on the nasal side while on the oral side the epithelium is thinner and stratified. The staining of the basement membrane region is indistinct.The levator veli palatini muscles can be seen developing within the soft palate shelves, lateral to the seam. Even more laterally, at the base of the pterygoid hamulus, the tensor veli palatini muscles are developing and in this specimen appear very defined and robust. Key: lvp ? levator veli palatini, ns ? nasal septum, ppm ? palatine process of the maxilla, t ? tongue, tvp ? tensor veli palatini. Scale bar for A,B,C =300?m; A?, B?, C?, =100?m.   	 ? 36	 ?3.2.2 57-day specimens have completed hard but not soft palate closure 	 ?By	 ?57-??days,	 ?the	 ?fetal	 ?period	 ?has	 ?begun.	 ?During	 ?the	 ?fetal	 ?period	 ?the	 ?tissues	 ?and	 ?organs	 ?of	 ?the	 ?body	 ?mature,	 ?and	 ?the	 ?fetus	 ?experiences	 ?rapid	 ?growth	 ?(Sadler	 ?and	 ?Langman,	 ?2010b).	 ?In	 ?my	 ?study	 ?there	 ?were	 ?four	 ?57-??day	 ?specimens	 ?collected	 ?(Fig	 ?3.3	 ?and	 ?3.4	 ?and	 ?data	 ?not	 ?shown	 ?H10142	 ?and	 ?H10458).	 ?The	 ?four	 ?57-??day	 ?specimens	 ?examined	 ?had	 ?fairly	 ?similar	 ?development	 ?in	 ?that	 ?all	 ?had	 ?completed	 ?hard	 ?palate	 ?fusion	 ?but	 ?the	 ?uvula	 ?had	 ?not	 ?formed	 ?(Tables	 ?3.1,	 ?3.3).	 ?	 ?In	 ?all	 ?specimens	 ?a	 ?few	 ?epithelial	 ?remnants	 ?of	 ?the	 ?epithelial	 ?seam	 ?are	 ?present	 ?(Fig.	 ?3.3,	 ?Fig	 ?3.4).	 ?Surprisingly,	 ?the	 ?57-??day	 ?specimens	 ?exhibited	 ?less	 ?advanced	 ?soft	 ?palate	 ?development	 ?than	 ?the	 ?more	 ?mature	 ?54-??day	 ?specimen	 ?(Fig	 ?3.1).	 ?This	 ?suggests	 ?that	 ?the	 ?54-??day	 ?embryo	 ?was	 ?precocious.	 ?In	 ?one	 ?57-??day	 ?specimen	 ?there	 ?is	 ?almost	 ?a	 ?continuous	 ?midline	 ?seam	 ?in	 ?the	 ?hard	 ?palate	 ?(Fig.	 ?3.3A).	 ?The	 ?middle	 ?of	 ?the	 ?soft	 ?palate	 ?(Fig.	 ?3.3B)	 ?has	 ?an	 ?intact	 ?midline	 ?seam,	 ?with	 ?a	 ?prominent	 ?epithelial	 ?thickening	 ?or	 ?triangle	 ?on	 ?the	 ?oral	 ?side.	 ?Condensations	 ?in	 ?the	 ?middle	 ?of	 ?the	 ?shelves	 ?may	 ?indicate	 ?presence	 ?of	 ?the	 ?early	 ?palatine	 ?aponeurosis.	 ?Lateral	 ?to	 ?this,	 ?areas	 ?of	 ?the	 ?developing	 ?tensor	 ?veli	 ?palatine	 ?can	 ?be	 ?seen	 ?directly	 ?below	 ?the	 ?palatine	 ?bones	 ?(Fig.	 ?3.3C,C?).	 ?	 ?	 ?The	 ?most	 ?posterior	 ?part	 ?of	 ?the	 ?soft	 ?palate	 ?is	 ?not	 ?contacting	 ?in	 ?the	 ?midline	 ?(Fig.	 ?3.3C,C?).	 ?	 ?In	 ?a	 ?second	 ?57-??day	 ?specimen	 ?(Fig.	 ?3.4)	 ?also	 ?has	 ?a	 ?continuous	 ?midline	 ?seam	 ?through	 ?the	 ?hard	 ?palate	 ?(Fig.	 ?3.3A).	 ?The	 ?seam	 ?increases	 ?in	 ?thickness	 ?as	 ?it	 ?transitions	 ?from	 ?the	 ?superior	 ?nasal	 ?side	 ?of	 ?the	 ?palate	 ?to	 ?the	 ?inferior	 ?oral	 ?side	 ?of	 ?the	 ?palate.	 ?The	 ?anterior	 ?soft	 ?palate	 ?section	 ?(Fig.	 ?3.4B)	 ?has	 ?a	 ?prominent	 ?midline	 ?seam,	 ?with	 ?small	 ?epithelial	 ?islands	 ?lateral	 ?to	 ?the	 ?midline	 ?(Fig.	 ? 	 ? 37	 ?3.4B?).	 ?Generally,	 ?the	 ?epithelial	 ?seam	 ?in	 ?the	 ?soft	 ?palate	 ?is	 ?thicker	 ?and	 ?less	 ?uniform	 ?than	 ?that	 ?seen	 ?in	 ?the	 ?hard	 ?palate	 ?	 ?(Fig.	 ?3.3B).	 ?	 ?	 ?	 ?In	 ?the	 ?posterior	 ?soft	 ?palate	 ?(Fig.	 ?3.4C),	 ?there	 ?is	 ?contact	 ?between	 ?the	 ?right	 ?and	 ?left	 ?palatal	 ?shelves	 ?but	 ?a	 ?thin	 ?epithelial	 ?bridge	 ?persists	 ?between	 ?the	 ?two	 ?sides.	 ?The	 ?oral	 ?surface	 ?epithelium	 ?is	 ?consistently	 ?much	 ?thicker	 ?and	 ?more	 ?irregular	 ?in	 ?appearance	 ?than	 ?that	 ?of	 ?the	 ?nasal	 ?surface.	 ?Although	 ?I	 ?did	 ?not	 ?use	 ?a	 ?specific	 ?stain	 ?for	 ?basement	 ?membrane	 ?it	 ?is	 ?generally	 ?harder	 ?to	 ?distinguish	 ?the	 ?basement	 ?membrane	 ?region	 ?from	 ?the	 ?underlying	 ?mesenchyme	 ?in	 ?the	 ?soft	 ?palate	 ?than	 ?in	 ?the	 ?hard	 ?palate	 ?(Fig.	 ?3.3B?,C?).	 ?The	 ?tensor	 ?veli	 ?palatini	 ?muscle	 ?can	 ?be	 ?seen	 ?on	 ?the	 ?right	 ?side	 ?below	 ?the	 ?palatine	 ?bone.	 ?In	 ?the	 ?terminal	 ?soft	 ?palate	 ?(Fig.	 ?3.4D),	 ?the	 ?two	 ?shelves	 ?have	 ?not	 ?yet	 ?contacted,	 ?and	 ?they	 ?are	 ?fairly	 ?small	 ?in	 ?size..	 ?In	 ?summary,	 ?at	 ?57-??days	 ?the	 ?soft	 ?palate	 ?has	 ?started	 ?to	 ?fuse	 ?but	 ?is	 ?incomplete	 ?in	 ?the	 ?area	 ?of	 ?the	 ?uvula.	 ?Importantly	 ?there	 ?is	 ?clear	 ?evidence	 ?for	 ?a	 ?midline	 ?epithelial	 ?seam	 ?in	 ?the	 ?soft	 ?palate	 ?at	 ?57-??days.	 ?The	 ?invasion	 ?of	 ?the	 ?tensor	 ?veli	 ?palatini	 ?has	 ?started	 ?and	 ?the	 ?aponeurosis	 ?is	 ?also	 ?beginning.	 ? 	 ? 38	 ?Figure	 ?3.3	 ?Development	 ?of	 ?the	 ?palate	 ?in	 ?a	 ?57-?day	 ?fetus.	 ?Frontal sections stained with hematoxylin and eosin,  At the posterior edge of the hard palate (A, A?), the shelves have fused and the darkly stained seam between the palatal shelves  is fully intact with no regions of degradation (arrows).  In the anterior soft palate (B, B?), the palatal shelves have also contacted and fused, the seam is breaking down, with multiple islands of epithelium (arrows). The palatine aponeurosis is seen condensing in both the right and left palatal shelves (black arrowheads). A small portion of the developing tensor veli palatini muscle is present in the right palatal shelf (white arrowhead).  In the posterior soft palate (C, C?), initial contact has been made between the palatal shelves. A bridge of epithelium represents the initial point of fusion between the shelves. Thick layers of epithelium (C?) can be seen on the oral side off the pre-fusion soft palate shelves. Note the indistinct staining of the epithelium compared to the mesenchyme.The aponeurosis is seen condensing in the right and left soft palate shelves (black arrowheads), and the tensor veli palatini is seen on the right side (white arrowhead) developing by the pterygoid hamulus.  In the terminal soft palate (D, D?), the soft palate shelves have not yet contacted. These shelves are extremely small and thin compared to the fused shelves, indicating that they still need to grow in order to complete development. The left tensor veli palatini (white arrowhead) is seen in this section, as are the right and left levator veli palatini muscles developing within the soft palatal shelves (black arrowheads). Key: ns ? nasal septum, pb ? palatine bone, ph ? pterygoid hamulus, ppm ? palatine process of the maxilla, t ? tongue, tvp ? tensor veli palatini. Scale bar =525 ?m for A,B,C,D; bar = 200 ?m for A?, B?, C?,D?.   	 ? 39	 ?	 ?Figure	 ?3.4	 ?	 ?Palatal	 ?morphogenesis	 ?in	 ?a	 ?second	 ?57-?day	 ?specimen	 ?Frontal sections of a second 57-day specimen stained with Hematoxylin and Eosin, At the posterior edge of the hard palate (A, A?), the palatal shelves are fully fused to each other and to the nasal septum. There is a bilayered seam present between the right and left palatal shelves, which is only in the initial stages of breaking down (arrows). There are small areas of epithelium the seam that appear to be more mesenchymal in appearance (black arrowheads).  In the anterior soft palate (B, B?), the soft palate shelves have contacted in the midline of the superior aspect of the shelves. The midline seam between the shelves is intact and appears of mostly uniform thickness. Inferior to the base of the seam, the soft palate shelves have not yet contacted and are still open. Early condensations of the left and right palatine aponeurosis can be seen in these anterior soft palate shelves (white arrowheads). The tensor veli palatini is the only palatine muscle that can be seen developing in this section. In the posterior soft palate, (C, C?), the section has torn through the shelves, but it appears that the shelves in this aspect of the soft palate had not yet contacted. Thick epithelium lining the shelves can be seen on the nasal side of the shelves. The oral side epithelium has mostly torn off this specimen. Key: ns ? nasal septum, pb ? palatine bone, ppm ? palatine process of the maxilla, t ? tongue.  Scale bar =525?m for A,B,C; bar = 200 ?m for A?, B?, C?.   	 ? 40	 ?3.2.3 59-70-day specimens have complete hard palates, variable soft palate development 	 ?The	 ?two	 ?59	 ?day	 ?specimens	 ?examined	 ?were	 ?greatly	 ?varied	 ?in	 ?their	 ?degree	 ?of	 ?development	 ?	 ?.	 ?One	 ?exhibits	 ?complete	 ?development	 ?of	 ?the	 ?soft	 ?palate	 ?and	 ?development	 ?of	 ?the	 ?aponeurosis	 ?(Table	 ?3.1,	 ?3.3,	 ?H9652),	 ?while	 ?the	 ?other	 ?has	 ?a	 ?developed	 ?hard	 ?palate	 ?but	 ?the	 ?soft	 ?palate	 ?is	 ?only	 ?partially	 ?formed	 ?similar	 ?to	 ?the	 ?57-??day	 ?specimens	 ?(Fig.	 ?3.7A-??C,	 ?	 ?H10593).	 ?The	 ?more	 ?advanced	 ?59-??day	 ?specimen	 ?is	 ?similar	 ?to	 ?the	 ?70-??day	 ?specimens	 ?leading	 ?me	 ?to	 ?conclude	 ?that	 ?this	 ?particular	 ?specimen	 ?is	 ?not	 ?representative.	 ?	 ?Further	 ?evidence	 ?to	 ?say	 ?that	 ?the	 ?typical	 ?59	 ?day	 ?fetus	 ?has	 ?a	 ?partially	 ?fused	 ?palate	 ?comes	 ?from	 ?the	 ?examination	 ?of	 ?64	 ?and	 ?67-??day	 ?specimens.	 ?In	 ?the	 ?64-??day	 ?specimens	 ?a	 ?few	 ?epithelial	 ?remnants	 ?of	 ?the	 ?epithelial	 ?seam	 ?in	 ?the	 ?hard	 ?palate	 ?are	 ?present	 ?(Fig.	 ?3.4A).	 ?The	 ?horizontal	 ?processes	 ?of	 ?the	 ?palatine	 ?bones	 ?are	 ?extending	 ?towards	 ?the	 ?midline	 ?and	 ?the	 ?nasal	 ?septum	 ?is	 ?continuous	 ?with	 ?the	 ?nasal	 ?side	 ?of	 ?the	 ?palatal	 ?shelves.	 ?The	 ?anterior	 ?and	 ?middle	 ?soft	 ?palate	 ?sections	 ?show	 ?complete	 ?degradation	 ?of	 ?the	 ?midline	 ?seam	 ?(Fig.	 ?3.5B,B?),	 ?however	 ?the	 ?seam	 ?is	 ?evident	 ?in	 ?posterior	 ?sections	 ?(Fig.	 ?3.5C,C?).	 ?The	 ?posterior	 ?seam	 ?consists	 ?of	 ?thickened	 ?epithelium	 ?on	 ?the	 ?oral	 ?side.	 ?In	 ?the	 ?posterior	 ?sections,	 ?the	 ?mesenchymal	 ?condensations	 ?for	 ?the	 ?developing	 ?palatine	 ?aponeurosis	 ?are	 ?visible	 ?(Fig.	 ?3.4C).	 ?.	 ?The	 ?67-??day	 ?specimen	 ?(Fig	 ?3.7	 ?D-??F	 ?and	 ?Table	 ?3.1,	 ?3.3)	 ?has	 ?a	 ?fused	 ?soft	 ?palate	 ?with	 ?no	 ?seam	 ?present,	 ?until	 ?the	 ?posterior	 ?aspect	 ?which	 ?is	 ?still	 ?unfused.	 ?By	 ?this	 ?point,	 ?the	 ?aponeurosis	 ?is	 ?more	 ?robust	 ? 	 ? 41	 ?in	 ?the	 ?anterior	 ?and	 ?mid	 ?soft	 ?palate	 ?(Fig	 ?3.7	 ?D,E	 ?insets)	 ?and	 ?has	 ?almost	 ?connected	 ?in	 ?the	 ?midline.	 ?	 ? 	 ? 42	 ?	 ?	 ?Figure	 ?	 ?3.5.	 ?Palatal	 ?morphogenesis	 ?of	 ?a	 ?64-?day	 ?fetus.	 ?Frontal sections through the palate stained with Picrosirius Red and Alcian Blue, This specimen was tiled so that the right side is more anterior than the left side.  At the posterior edge of the hard palate (A, A?), the palatal shelves have fused. There are remnants of a midline seam in this region of the hard palate. The palatine bone is extending in to the hard palate on the right side.  The anterior soft palate (B, B?) is fused in the midline, with no remnants of midline seam remaining. The epithelium on the inferior aspect of the midline of the palate is thick but regular in its appearance. The palatine aponeurosis and levator veli palatini are visible in the left side of the palate. The sectioning bias of this specimen is evident in the posterior soft palate (C, C?), as the right side of the soft palate displays a full right shelf, that has contacted and fused with part of a left shelf, with a full midline seam present between the shelves (C?). The staining of the seam is very light and similar in colour to the mesenchymal stain.The aponeurosis is also present throughout the right shelf. On the lateral aspect of the left side, the more posterior developing palatal shelf is seen as a dome shaped outgrowth from the lateral pharyngeal wall. Cells of the developing palatopharyngeus muscle can be seen in this structure. Key: apo ? aponeurosis,  mes ? midline epithelial seam, ns ? nasal septum, pb ? palatine bone, ppm ? palatine process of the maxilla, tvp ? tensor veli palatini.  Scale bar in A=525?m applies to A,B,C; bar in A? = 200 ?m applies to A?, B?, C?. 	 ? 	 ? 43	 ?	 ?There	 ?were	 ?two	 ?70-??day	 ?specimens	 ?but	 ?unfortunately	 ?only	 ?one	 ?of	 ?these	 ?included	 ?the	 ?full	 ?soft	 ?palate	 ?(data	 ?not	 ?shown,	 ?H9705).	 ?The	 ?other	 ?was	 ?missing	 ?tissue	 ?posterior	 ?to	 ?the	 ?junction	 ?of	 ?the	 ?hard	 ?and	 ?soft	 ?palate	 ?and	 ?was	 ?uninformative	 ?(data	 ?not	 ?shown,	 ?H9832).	 ?	 ?The	 ?intact	 ?specimen	 ?had	 ?full	 ?fusion	 ?of	 ?the	 ?soft	 ?palate	 ?and	 ?only	 ?the	 ?uvula	 ?retained	 ?part	 ?of	 ?the	 ?epithelial	 ?seam.	 ?This	 ?specimen	 ?provided	 ?convincing	 ?evidence	 ?for	 ?a	 ?model	 ?of	 ?fusion	 ?of	 ?the	 ?entire	 ?soft	 ?palate	 ?from	 ?the	 ?anterior	 ?aspect	 ?to	 ?the	 ?tip	 ?of	 ?the	 ?uvula,	 ?contradicting	 ?a	 ?previous	 ?theory	 ?of	 ?fusion	 ?of	 ?the	 ?anterior	 ?2/3	 ?of	 ?the	 ?soft	 ?palate	 ?and	 ?merging	 ?of	 ?the	 ?uvula	 ?(Poswillo,	 ?1974).	 ?Interestingly,	 ?the	 ?hard	 ?palate	 ?retains	 ?numerous	 ?midline	 ?epithelial	 ?islands	 ?well	 ?into	 ?the	 ?fetal	 ?period	 ?and	 ?beyond	 ?whereas	 ?the	 ?seam	 ?in	 ?the	 ?soft	 ?palate	 ?is	 ?much	 ?more	 ?transient.	 ?	 ?	 ?3.2.4 74-day specimen has a fully developed soft palate and aponeurosis 	 ?The	 ?only	 ?74-??day	 ?specimen	 ?obtained	 ?was	 ?sectioned	 ?in	 ?the	 ?coronal	 ?plane	 ?or	 ?in	 ?the	 ?same	 ?plane	 ?as	 ?the	 ?palate	 ?itself	 ?(data	 ?not	 ?shown,	 ?H9819,	 ?Table	 ?	 ?This	 ?is	 ?the	 ?only	 ?specimen	 ?in	 ?my	 ?study	 ?in	 ?which	 ?the,	 ?soft	 ?palate	 ?is	 ?fully	 ?formed	 ?as	 ?shown	 ?by	 ?the	 ?complete	 ?absence	 ?of	 ?epithelial	 ?islands	 ?or	 ?a	 ?seam	 ?in	 ?the	 ?midline	 ?mesenchyme.The	 ?aponeurosis	 ?is	 ?continuous	 ?across	 ?the	 ?soft	 ?palate	 ?and	 ?the	 ?tensor	 ?veli	 ?palatini	 ?muscles	 ?have	 ?attached	 ?to	 ?its	 ?distal	 ?ends.	 ?.	 ?As	 ?noted	 ?for	 ?the	 ?70-??day	 ?specimens,	 ?epithelial	 ?pearls	 ?are	 ?present	 ?in	 ?the	 ?midline	 ?of	 ?the	 ?hard	 ?palate.	 ?	 ?Thus	 ?soft	 ?palate	 ?fusion	 ?is	 ?complete	 ?between	 ?70	 ?and	 ?74-??days.	 ? 	 ? 44	 ?3.2.5 3-D reconstructions confirm an epithelial seam is present in the soft palate 	 ?In	 ?order	 ?to	 ?clarify	 ?the	 ?relationship	 ?of	 ?a	 ?seam	 ?to	 ?the	 ?soft	 ?and	 ?hard	 ?palate	 ?I	 ?reconstructed	 ?a	 ?variety	 ?of	 ?specimens	 ?ranging	 ?in	 ?age	 ?from	 ?54	 ?to	 ?74-??days	 ?(54-??days	 ?n	 ?=	 ?2,	 ?57-??days	 ?n	 ?=	 ?4,	 ?59	 ?days	 ?n	 ?=	 ?2,	 ?64-??days	 ?n	 ?=	 ?1,	 ?67-??days	 ?n	 ?=	 ?1,	 ?70-??days	 ?n	 ?=	 ?1,	 ?74-??days	 ?n	 ?=	 ?1).	 ?I	 ?traced	 ?outlines	 ?for	 ?the	 ?palatal	 ?shelves	 ?of	 ?the	 ?hard	 ?and	 ?soft	 ?palate,	 ?the	 ?nasal	 ?septum,	 ?the	 ?palatine	 ?process	 ?of	 ?the	 ?maxilla	 ?and	 ?palatine	 ?bones,	 ?the	 ?greater	 ?palatine	 ?nerve,	 ?and	 ?the	 ?midline	 ?seam,	 ?and	 ?seam	 ?remnants	 ?when	 ?present.	 ?	 ?In	 ?the	 ?54-??day	 ?specimen,	 ?a	 ?midline	 ?seam	 ?is	 ?present	 ?in	 ?the	 ?entire	 ?soft	 ?palate	 ?ending	 ?where	 ?the	 ?palate	 ?has	 ?not	 ?yet	 ?fused	 ?(Fig	 ?3.6A,B).	 ?In	 ?the	 ?posterior	 ?aspect	 ?of	 ?the	 ?unfused	 ?soft	 ?palate,	 ?the	 ?shelves	 ?are	 ?diminutive	 ?in	 ?length	 ?and	 ?have	 ?a	 ?rounder,	 ?mushroom	 ?shape	 ?than	 ?the	 ?actively	 ?fusing	 ?parts	 ?of	 ?the	 ?shelf.	 ?These	 ?dome-??shaped	 ?shelves	 ?appear	 ?tethered	 ?to	 ?the	 ?lateral	 ?pharyngeal	 ?wall,	 ?and	 ?will	 ?have	 ?to	 ?reorient	 ?and	 ?grow	 ?outwards	 ?towards	 ?the	 ?midline	 ?before	 ?these	 ?soft	 ?palate	 ?shelves	 ?can	 ?touch	 ?in	 ?the	 ?midline.	 ?	 ?A	 ?midline	 ?epithelial	 ?seam	 ?was	 ?also	 ?present	 ?throughout	 ?the	 ?hard	 ?palate	 ?and	 ?into	 ?the	 ?soft	 ?palate	 ?in	 ?the	 ?57-??day	 ?specimens	 ?traced	 ?(Fig	 ?3.6C,D	 ?and	 ?data	 ?not	 ?shown	 ?H9517,	 ?H10142,	 ?H10458).	 ?The	 ?terminal	 ?portions	 ?of	 ?the	 ?soft	 ?palate	 ?shelves	 ?in	 ?these	 ?57	 ?day	 ?specimens	 ?start	 ?to	 ?take	 ?on	 ?a	 ?different	 ?shape,	 ?they	 ?have	 ?lost	 ?the	 ?dome-??like	 ?shape	 ?projecting	 ?from	 ?the	 ?pharyngeal	 ?walls	 ?and	 ?become	 ?more	 ?thin	 ?and	 ?tapered	 ?as	 ?they	 ?elongate	 ?toward	 ?the	 ?midline.	 ?By	 ?the	 ?64th	 ?day,	 ?the	 ?medial	 ?epithelial	 ?seam	 ?(Fig	 ?3.6E,F)	 ?has	 ?degraded.	 ?I	 ?also	 ?traced	 ?the	 ?outline	 ?of	 ?the	 ?aponeurosis	 ?because	 ?the	 ?condensations	 ?were	 ?clearly	 ?visible	 ?in	 ?the	 ?64-??day	 ?specimen.	 ?In	 ?the	 ?70-??day	 ?specimen,	 ?the	 ?shape	 ?of	 ?the	 ?soft	 ?palate	 ? 	 ? 45	 ?has	 ?changed	 ?as	 ?the	 ?uvula	 ?is	 ?developing	 ?(Fig	 ?3.6G,	 ?H9832).	 ?There	 ?is	 ?an	 ?abrupt	 ?change	 ?from	 ?a	 ?horizontal	 ?to	 ?vertical	 ?orientation	 ?of	 ?the	 ?soft	 ?palate	 ?of	 ?this	 ?70-??day	 ?specimen	 ?at	 ?the	 ?posterior	 ?aspect	 ?and	 ?a	 ?midline	 ?seam	 ?can	 ?be	 ?seen	 ?only	 ?in	 ?the	 ?developing	 ?uvula	 ?(Fig	 ?3.6G,	 ?H9832).	 ?	 ?By	 ?74-??days	 ?(data	 ?not	 ?shown,	 ?H9819),	 ?the	 ?hard	 ?and	 ?soft	 ?palate	 ?morphology	 ?is	 ?fully	 ?formed,	 ?including	 ?the	 ?uvula.	 ?There	 ?is	 ?a	 ?continuous	 ?aponeurosis	 ?across	 ?the	 ?soft	 ?palate.	 ?No	 ?seam	 ?remains,	 ?however	 ?a	 ?few	 ?epithelial	 ?pearls	 ?are	 ?present	 ?in	 ?the	 ?midline	 ?of	 ?the	 ?hard	 ?palate	 ?(data	 ?not	 ?shown,	 ?H9819).	 ?	 ? 	 ? 46	 ?Figure	 ?	 ?3.6.	 ?3D	 ?reconstructions	 ?of	 ?the	 ?hard	 ?palate-?soft	 ?palate	 ?junctionAll specimens are shown from a palatal and a posterior view, with the exception of the 70-day specimen (G), which is displayed from a sagittal view. White arrowheads point to the hard palate-soft palate junction . In the 54-day specimen (A, B), the seam is present through the entire fused soft palate (red). The unfused soft palate shelves in this reconstruction are significantly smaller in volume than the fused shelves, indicating that they may need to increase in volume before they can contact and fuse.   This is also demonstrated from the posterior view (B), where the dome shape of these unfused shelves can be clearly seen. In the 57-day specimen (C, D), the midline seam is proliferating through the entire fused hard and soft palate (blue). The unfused shelves in this specimen are larger in volume and closer to contacting in the midline(C, D) than the shelves of the 54-day specimen (A, B). ). The 57 day shelves also have a different shape, they are more tapered in appearance.   In the 64-day specimen (E, F), the seam in the hard palate and initial soft palate is still present (blue), then in the anterior soft palate it has disappeared, then reappears in the middle to posterior fusing soft palate. The aponeurosis (purple) can also be seen migrating in to the left palatal shelf (F).  In the sagittal reconstruction of the 70-day specimen (G) we can see the change in location of the soft palate (red) from the hard palate (orange) as the soft palate hangs lower than the hard palate, representing the transition from a horizontal to a more vertically-oriented structure.  	 ? 	 ? 47	 ?	 ? 3.2.6 Palatine aponeurosis condenses near the tensor veli palatini between 59 and 67d 	 ?By	 ?59	 ?days,	 ?the	 ?majority	 ?of	 ?the	 ?muscles	 ?of	 ?the	 ?fetal	 ?craniofacial	 ?complex	 ?have	 ?begun	 ?to	 ?develop.	 ?In	 ?the	 ?59	 ?day	 ?specimen	 ?stained	 ?with	 ?MF20	 ?antibody	 ?(Fig	 ?3.7A,B,C),	 ?the	 ?tongue	 ?muscle	 ?is	 ?prominent	 ?and	 ?the	 ?medial	 ?pterygoid	 ?and	 ?the	 ?masseter	 ?muscle	 ?can	 ?be	 ?seen	 ?lying	 ?on	 ?either	 ?side	 ?of	 ?Meckel?s	 ?cartilage	 ?(Fig	 ?3.7A).	 ?	 ?At	 ?a	 ?more	 ?posterior	 ?location	 ?in	 ?the	 ?59	 ?day	 ?specimen	 ?(Fig	 ?3.7B),	 ?the	 ?tensor	 ?veli	 ?palatini	 ?muscle	 ?can	 ?be	 ?clearly	 ?seen	 ?in	 ?its	 ?position	 ?lateral	 ?to	 ?the	 ?developing	 ?pterygoid	 ?hamulus,	 ?the	 ?levator	 ?veli	 ?palatini	 ?is	 ?developing	 ?within	 ?the	 ?body	 ?of	 ?the	 ?soft	 ?palate	 ?and	 ?early	 ?signs	 ?of	 ?the	 ?palatoglossus	 ?muscle	 ?can	 ?be	 ?seen	 ?sitting	 ?more	 ?inferiorly	 ?at	 ?the	 ?base	 ?of	 ?the	 ?tongue.	 ?The	 ?palatopharyngeus	 ?can	 ?also	 ?be	 ?seen	 ?in	 ?its	 ?early	 ?stage	 ?of	 ?development	 ?(Fig	 ?3.7C).	 ?By	 ?67-??days	 ?(Fig.	 ?3.7	 ?D,D?,E,E?,F,F?)	 ?the	 ?muscles	 ?are	 ?more	 ?robust	 ?and	 ?organized.	 ?The	 ?condensation	 ?of	 ?the	 ?developing	 ?aponeurosis	 ?can	 ?be	 ?seen	 ?(Fig	 ?3.7D,E	 ?insets).	 ?The	 ?palatoglossus	 ?is	 ?migrating	 ?superiorly	 ?towards	 ?the	 ?soft	 ?palate	 ?(Fig	 ?3.7E)	 ?and	 ?the	 ?palatopharyngeus	 ?is	 ?increasing	 ?in	 ?mass	 ?(Fig	 ?3.7F,	 ?F?).	 ?	 ?By	 ?day	 ?74	 ?(data	 ?not	 ?shown,	 ?H9819),	 ?the	 ?aponeurosis	 ?has	 ?migrated	 ?across	 ?the	 ?soft	 ?palate	 ?and	 ?has	 ?attached	 ?to	 ?the	 ?tensor	 ?veli	 ?palatini	 ?to	 ?develop	 ?its	 ?postnatal	 ?morphology.	 ?	 ? 	 ? 48	 ?	 ?	 ?Figure	 ?3.7.	 ?Palatal	 ?muscle	 ?development	 ?in	 ?59	 ?and	 ?67-?day	 ?fetuses.	 ? Frontal sections of 59 and 67-day specimens through the soft palate. Muscle cells stained with MF20 antibody.  Specimens counterstained with either hematoxylin and eosin (A, C) or Picrosirius Red and Alcian Blue.  In the 59 day specimen (A-C),  many muscles can be seen developing. In the anterior soft palate (A), the aponeurosis is seen in the palatal shelves, the tongue muscle is evident and the medial pterygoid and masseter muscles are visible developing on the medial and lateral aspects respectively of the developing mandible and Meckels cartilage. In the middle of the soft palate (B), the shelves have not yet fused. The tvp is visible on the lateral side of the pterygoid hamulus and the lvp is developing in the middle of the palatal shelves. The palatoglossus muscles are also visible. In the posterior soft palate (C), the palatopharyngeus muscle cells are starting to form. In the 67-day specimen, the lvp muscle is more defined in the anterior and middle soft palate (D, D?, E, E?). The palatine aponeurosis is thicker and more condensed and is almost continuous across the middle of the palate (D, E insets). The palatopharyngeus muscle is becoming larger and more organized (F, F?) than in the 59 day specimen (C). Key: Key: apo ? palatine aponeurosis, lvp ? levator veli palatini, ma ? masseter, M ? Meckels cartilage, mp - medial pterygoid, pg ? palatoglossus, pp-palatopharyngeus, tvp ? tensor veli palatini, t ? tongue, scale bar in A =525?m, applies to A,B,C,D,E,F; scale bar in D? =200?m, applies to D?,E?,F?.	 ? 	 ? 49	 ?Chapter 4 ?Discussion 	 ? 4.1.  Revised model of soft palate development  The results of my study have shown that fusion of the palatal shelves is the main mechanism by which the soft palate forms. As palatal shelf fusion occurs in an anterior to posterior direction, the soft palate shelves grow out from the lateral wall of the pharynx towards the midline and contact to form a midline epithelial seam (Fig 4.1A-C). My study advocates that the process of fusion is occurring throughout the soft palate into the uvula, as one 70-day specimen (H9705) showed the presence of a midline epithelial seam extending into the tip of the uvula. This contradicts the theory proposed by Burdi and Faist (Burdi and Faist, 1967), but clear cut evidence of a midline seam in the soft palate supports fusion as the prevailing method of soft palate development. The unanticipated finding in my study was that are independent rates of development being followed in the hard and soft palate. The hard palate seam fusion progresses rather slowly from anterior to posterior. Thus I often encountered robust seams in the posterior hard palate. Yet the soft palate seam forms and degrades very rapidly. Thus there are specimens in which the anterior soft palate seam is degrading or completely removed at the same time when the posterior hard palate seam is largely intact. This is best seen in the 64 day specimen (Fig. 3.5). The hard palate seam can persist from the time of fusion to birth, a 34 week time period. The soft palate seam was only seen for a 16 day time period, from 54-70 days.  It will be important to expand my sample to confirm this novel idea.   	 ? 50	 ?	 ?	 ?Figure	 ?	 ?4.1.	 ?The	 ?mechanism	 ?of	 ?palate	 ?closure	 ?is	 ?fusion	 ?in	 ?both	 ?the	 ?hard	 ?and	 ?soft	 ?palate. 	 ?	 ?This diagrammatic representation is an update of the diagram included in the 1967 study on palatal morphogenesis by Burdi and Faist.  It illustrates my proposed method of soft palate development. Hard palate development is unchanged from the previous diagram with outgrowth of the palatal shelves towards the midline and primary palate. The gradients indicate the maturity of the seam. The darker the colour the more mature.  There is a disconnect between the rate of maturation of the seam in the hard and soft palates such that the two structures appear to be forming almost independently of each other. The soft palate seam may be completely removed at the same time as there is a seam present in the posterior hard palate. (A), contact occurs and the shelves fuse in an anterior-posterior direction (B,). As the hard palate is fusing, the soft palate shelves are growing outwards from the lateral pharyngeal wall and contacting in the midline (B), after which point fusion occurs and the midline epithelial seam is degraded (C).  The inserts show the differences in the appearance of the hard vs soft palate epithelium at the point of fusion. The hard palate epithelium (A?) is highly organized, displays an intact basement membrane and is keratinized, based on the contrast in epithelial and mesenchymal cell staining. The soft palate epithelium (B?) is disorganized and bulky, with clumping of cells and a fragmented basement membrane. There is also poor keratinization of the soft palate epithelial cells.    	 ? 51	 ? It is important to note the differences in the appearance of the epithelium between the hard and soft palate during fusion (Fig 4.1A?, B?). The epithelium of the hard palate is regular and uniform in appearance. It is approximately 2-3 cells thick, stains darkly with H&E compared to the mesenchymal cells, and has distinct staining compared to the mesenchyme which suggests an intact basement membrane. Conversely the soft palate epithelium is very disorganized in appearance. It is much bulkier and less regular in appearance than the hard palate, ranging from 2-20 cells thick. With both H&E and Picrosirius Red staining, the epithelial cells only stain very lightly and are difficult to distinguish from the mesenchyme, due to poor keratinization of the soft palate epithelium.  The basement membrane in the soft palate may be fragmented and difficult to identify. Future studies should examine more closely the different types of keratins in hard and soft palate seam as well as the presence of basement membrane.  These differences are likely due to the different embryonic origins of the two structures. While no direct evidence exists from experimental studies, some anatomists have postulated that the hard palate epithelium arises from ectoderm from the stomodeum, whereas the soft palate originates from foregut endoderm (Singh, 2005).  In a study on the regulation of Shh in mice found there was an enhancer that drove expression in the soft palate and pharyngeal epithelium. When this enhancer was deleted the soft palate was shortened, the epiglottis and posterior tongue were abnormal. It was not possible to separate out the regulation of gene expression in the soft palate from that of the pharynx which supports a common embryonic origin (Sagai et al., 2009). These different origins may also be responsible for the temporal differences in seam degradation between the hard and soft palates.   	 ? 52	 ?4.2. Comparative Anatomy - Do other mammals have a seam in the soft palate? 	 ?In	 ?this	 ?section	 ?I	 ?will	 ?provide	 ?a	 ?broader	 ?perspective	 ?on	 ?how	 ?representative	 ?human	 ?soft	 ?palate	 ?fusion	 ?is	 ?in	 ?relation	 ?to	 ?other	 ?mammals.	 ?	 ?During	 ?evolution	 ?of	 ?mammals	 ?the	 ?assumption	 ?is	 ?that	 ?developmental	 ?mechanisms	 ?would	 ?be	 ?conserved.	 ?Certainly	 ?it	 ?is	 ?true	 ?that	 ?all	 ?mammals	 ?pass	 ?through	 ?a	 ?stage	 ?of	 ?palatal	 ?shelf	 ?morphogenesis,	 ?reorientation	 ?and	 ?fusion	 ?but	 ?whether	 ?or	 ?not	 ?a	 ?seam	 ?is	 ?present	 ?in	 ?the	 ?soft	 ?palate	 ?has	 ?not	 ?received	 ?as	 ?much	 ?attention.	 ?	 ?In	 ?the	 ?mouse	 ?it	 ?is	 ?well	 ?known	 ?that	 ?the	 ?soft	 ?palate	 ?in	 ?mice	 ?develops	 ?by	 ?fusion,	 ?not	 ?merging	 ?(Smiley,	 ?1975)	 ?.	 ?Ultrastructural	 ?studies	 ?were	 ?performed	 ?on	 ?hamster	 ?palates	 ?(Shah	 ?and	 ?Chaudhry,	 ?1974).	 ?Palatal	 ?closure	 ?occurred	 ?in	 ?hamsters	 ?between	 ?days	 ?12	 ?and	 ?13	 ?of	 ?gestation,	 ?with	 ?soft	 ?palate	 ?closure	 ?occurring	 ?over	 ?a	 ?four	 ?hour	 ?time	 ?period	 ?during	 ?these	 ?days.	 ?Prior	 ?to	 ?fusion,	 ?the	 ?medial	 ?edge	 ?epithelia	 ?of	 ?the	 ?shelves	 ?appeared	 ?thickened.	 ?Fusion	 ?was	 ?seen	 ?between	 ?soft	 ?palate	 ?shelves	 ?via	 ?desmosomal	 ?attachments,	 ?creating	 ?a	 ?midline	 ?epithelial	 ?seam	 ?with	 ?basal	 ?lamina	 ?on	 ?either	 ?side.	 ?Removal	 ?of	 ?the	 ?seam	 ?was	 ?observed	 ?by	 ?apoptosis,	 ?exfoliation	 ?and	 ?migration	 ?of	 ?epithelial	 ?cells	 ?(Shah	 ?and	 ?Chaudhry,	 ?1974).	 ?	 ?	 ?Rather	 ?surprisingly,	 ?no	 ?rat	 ?studies	 ?have	 ?looked	 ?at	 ?an	 ?exact	 ?mechanism	 ?of	 ?soft	 ?palate	 ?development,	 ?however	 ?it	 ?has	 ?been	 ?shown	 ?that	 ?there	 ?is	 ?a	 ?higher	 ?rate	 ?of	 ?epithelial	 ?cell	 ?renewal	 ?in	 ?the	 ?soft	 ?palate	 ?of	 ?rats	 ?versus	 ?the	 ?hard	 ?palate,	 ?which	 ?may	 ?indicate	 ?why	 ?a	 ?midline	 ?epithelial	 ?seam	 ?would	 ?break	 ?down	 ?faster	 ?in	 ?the	 ?soft	 ?palate	 ?than	 ?the	 ?hard	 ?palate	 ?(Hayward,	 ?1973;	 ?Hayward	 ?et	 ?al.,	 ?1973).	 ?	 ? 	 ? 53	 ?There	 ?is	 ?a	 ?single	 ?study	 ?performed	 ?on	 ?non-??human	 ?primates.	 ?In	 ?this	 ?study	 ?37	 ?baboon	 ?embryos	 ?were	 ?analyzed	 ?between	 ?30	 ?and	 ?64-??days	 ?(estimated)	 ?post	 ?conception	 ?(Bollert	 ?and	 ?Hendrickx,	 ?1971).	 ?	 ?In	 ?the	 ?53	 ?day	 ?and	 ?older	 ?specimens,	 ?the	 ?soft	 ?palates	 ?were	 ?formed	 ?with	 ?the	 ?exception	 ?of	 ?the	 ?uvula,	 ?which	 ?was	 ?still	 ?not	 ?formed	 ?in	 ?the	 ?oldest	 ?64-??day	 ?specimens.	 ?Upon	 ?inspection	 ?of	 ?these	 ?specimens,	 ?no	 ?epithelial	 ?remnants	 ?were	 ?seen	 ?in	 ?the	 ?soft	 ?palate,	 ?so	 ?the	 ?authors	 ?concluded	 ?that	 ?the	 ?soft	 ?palate	 ?must	 ?form	 ?by	 ?merging,	 ?however	 ?this	 ?may	 ?not	 ?actually	 ?be	 ?the	 ?mechanism	 ?of	 ?baboon	 ?soft	 ?palate	 ?development.	 ?	 ?Taken	 ?together,	 ?the	 ?evidence	 ?from	 ?mouse	 ?and	 ?hamster	 ?suggests	 ?that	 ?the	 ?most	 ?likely	 ?mechanism	 ?of	 ?soft	 ?palate	 ?closure	 ?in	 ?mammals	 ?is	 ?fusion.	 ?In	 ?other	 ?words	 ?it	 ?is	 ?hard	 ?to	 ?imagine	 ?that	 ?a	 ?different	 ?mechanism	 ?of	 ?palatogenesis	 ?of	 ?the	 ?soft	 ?palate	 ?would	 ?evolve	 ?in	 ?primates	 ?and	 ?humans.	 ?	 ?	 ?4.3. Variability of palate development in human fetuses  In my study, there was a degree of variability seen amongst the specimens, in particular two precocious specimens at 54 and 59 days had palates developed beyond those of older specimens (Table 3.1, 3.2). There are many different factors that could contribute to the variability.  Variations in form and development are intrinsic to all humans. Only in monozygotic twins would we be able to eliminate the genetic variability of development. Maternal factors also play a role in palatogenesis. Maternal smoking and amount of folic acid intake have both been shown to contribute to orofacial clefting in children (Butali et al., 2013). Certain medications such as cortisone and phenytoin also increase clefting rates (Carinci et al., 2007). Low amniotic fluid levels alter the  	 ? 54	 ?environment in which an embryo is developing.  Sex differences and racial variations also exist in embryo size and timing of development (O'Rahilly and Muller, 2010).  Males tend to have secondary palates that orient from vertical to horizontal earlier than females (Burdi and Silvey 1969), and the sex was not reported for any of the specimens collected in my study.  Palate development takes place over a period of weeks (from weeks 6-12 postconception), thus it is normal to expect some degree of variation in different specimens.  There was a 16 day window in my specimens in which a seam was visible: from days 54-70. Thus specimens observed have to be old enough that the soft palate shelves are in contact (ie 54 days and older), but young enough that the aponeurosis is not fully formed. In the 70 day specimen the only remaining soft palate seam was at the apex of the uvula.  Despite the intrinsic variability in development, is important to make every effort to be as accurate and rigorous as possible when dating the age of the human specimens. The criteria utilized at the University of Washington, where the tissue for this study was obtained, includes estimations from prenatal intakes, and the external measurements: foot length, Streeter?s stage and crown-rump length. Burdi and Faist only used crown-rump length and age in weeks to determine fetal age. The more factors utilized, the more accurate age dating of a specimen can be, however the information obtained from the University of Washington allowed me to directly compare the crown-rump lengths of my specimens with those of Burdi and Faist (Table 3.3). The increased range of crown rump lengths during development of the soft palate allowed me to see midline epithelial seams in the soft palate from the anterior aspect all the way to the uvula during various stages of development.   	 ? 55	 ?Overall, the specimens analyzed by Burdi and Faist followed the same general developmental milestones as my specimens, with some variability as well. Their specimens had initial secondary palate closure between 29 and 37mm crown-rump length. Complete soft palate closure was only seen in Burdi and Faist?s oldest specimen, measuring 75mm crown-rump length. My oldest specimen was also 75mm, and was also the only specimen in my collection that exhibited complete palatogenesis of the hard and soft palate.   4.4. Animal models give insights into the genes that are required for soft palate morphogenesis 	 ?There	 ?are	 ?hundreds	 ?of	 ?mouse	 ?models	 ?with	 ?cleft	 ?secondary	 ?palate	 ?but	 ?only	 ?a	 ?handful	 ?	 ?have	 ?a	 ?true	 ?submucous	 ?cleft.	 ?The	 ?Osr2-??IresCre;	 ?Bmpr1a	 ?fl/fl	 ?(Bone	 ?Morphogenetic	 ?Protein	 ?Receptor	 ?1a)	 ?(Baek	 ?et	 ?al.,	 ?2011)	 ?mouse	 ?is	 ?a	 ?mutant	 ?which	 ?displays	 ?inactivation	 ?of	 ?Bmpr1a	 ?in	 ?the	 ?palatal	 ?mesenchyme,	 ?resulting	 ?in	 ?a	 ?submucous	 ?cleft	 ?of	 ?the	 ?anterior	 ?soft	 ?palate.	 ?	 ?The	 ?phenotype	 ?of	 ?this	 ?mutant	 ?also	 ?included	 ?a	 ?gap	 ?between	 ?the	 ?primary	 ?and	 ?secondary	 ?palate	 ?and	 ?an	 ?irregular	 ?appearance	 ?of	 ?the	 ?palatal	 ?rugae.	 ?There	 ?was	 ?a	 ?lack	 ?of	 ?condensation	 ?of	 ?mesenchyme	 ?and	 ?agenesis	 ?of	 ?the	 ?palatine	 ?process	 ?of	 ?the	 ?maxilla.	 ?	 ?The	 ?palatal	 ?process	 ?of	 ?the	 ?palatine	 ?bone	 ?also	 ?has	 ?a	 ?decrease	 ?in	 ?osteogenesis	 ?and	 ?was	 ?diminutive	 ?in	 ?size.	 ?Bmpr1a	 ?mRNA	 ?is	 ?expressed	 ?in	 ?the	 ?mesenchyme	 ?of	 ?the	 ?medial	 ?nasal	 ?prominences	 ?and	 ?in	 ?the	 ?anterior	 ?half	 ?of	 ?the	 ?palatal	 ?shelves,	 ?with	 ?very	 ?little	 ?expression	 ?in	 ?posterior	 ?secondary	 ?palate.	 ?	 ?This	 ?mouse	 ?model	 ?indicates	 ?that	 ?Bmpr1a	 ?is	 ?required	 ?in	 ?the	 ?palatal	 ? 	 ? 56	 ?mesenchyme	 ?for	 ?normal	 ?palatogenesis	 ?and	 ?palatal	 ?bone	 ?development	 ?to	 ?occur.	 ?	 ?This	 ?submucous	 ?cleft	 ?phenotype	 ?of	 ?the	 ?Osr2-??IresCre;	 ?Bmpr1a	 ?fl/fl	 ?mouse	 ?arose	 ?from	 ?the	 ?combination	 ?of	 ?significantly	 ?reduced	 ?proliferation	 ?of	 ?the	 ?palatal	 ?mesenchymal	 ?and	 ?a	 ?lack	 ?of	 ?palatal	 ?bone	 ?support	 ?in	 ?the	 ?developing	 ?fetus.	 ?A	 ?signal	 ?that	 ?is	 ?directly	 ?involved	 ?in	 ?epithelial	 ?fusion	 ?of	 ?the	 ?palate	 ?is	 ?the	 ?protein,	 ?Transforming	 ?growth	 ?factor	 ?beta	 ?3	 ?(Tgf-???3).	 ?Tgf-???3	 ?null	 ?mouse	 ?mutants	 ?(Tgf-??3-??/-??)	 ?	 ?all	 ?exhibit	 ?some	 ?degree	 ?of	 ?clefting	 ?of	 ?the	 ?palate	 ?(Kaartinen	 ?et	 ?al.,	 ?1997;	 ?Kaartinen	 ?et	 ?al.,	 ?1995;	 ?Proetzel	 ?et	 ?al.,	 ?1995).	 ?The	 ?C57BL/6	 ?background	 ?exhibits	 ?the	 ?most	 ?severe	 ?clefting	 ?phenotype	 ?with	 ?50%	 ?of	 ?the	 ?mutants	 ?having	 ?complete	 ?clefts	 ?and	 ?the	 ?other	 ?50%	 ?having	 ?partial	 ?clefts	 ?(Cui	 ?et	 ?al.,	 ?2005).	 ?	 ?Tgf-???3	 ?regulates	 ?the	 ?disappearance	 ?of	 ?the	 ?medial	 ?edge	 ?epithelium	 ?in	 ?palatal	 ?shelf	 ?fusion,	 ?and	 ?knocking	 ?out	 ?this	 ?gene	 ?has	 ?a	 ?profound	 ?effect	 ?on	 ?shelf	 ?fusion.	 ?Tgf-???3	 ?directly	 ?regulates	 ?the	 ?phosphorylation	 ?of	 ?	 ?the	 ?transcription	 ?factor,	 ?Smad2,	 ?so	 ?when	 ?Smad2	 ?was	 ?overexpressed	 ?in	 ?the	 ?medial	 ?edge	 ?epithelium	 ?on	 ?a	 ?transgene,	 ?the	 ?cleft	 ?palate	 ?phenotype	 ?was	 ?rescued	 ?in	 ?Tgf-???3-??/-??(Cui	 ?et	 ?al.,	 ?2005).	 ?	 ?Of	 ?the	 ?6	 ?null	 ?mutant	 ?mice	 ?who	 ?carried	 ?the	 ?K14-??Smad2	 ?transgene	 ?(Tgf-???3-??/-??/K14-??Smad2	 ?),	 ?	 ?none	 ?had	 ?complete	 ?clefts,	 ?and	 ?3	 ?had	 ?fusion	 ?into	 ?the	 ?soft	 ?palate.	 ?In	 ?the	 ?5	 ?Tgf-???3-??/-??,	 ?4	 ?had	 ?complete	 ?clefts	 ?and	 ?only	 ?one	 ?had	 ?partial	 ?clefting	 ?(Cui	 ?et	 ?al.,	 ?2005).	 ?A	 ?study	 ?in	 ?which	 ?the	 ?receptor,	 ?Tgfbr2	 ?was	 ?conditionally	 ?deleted	 ?in	 ?the	 ?epithelium	 ?also	 ?generated	 ?submucous	 ?clefts	 ?in	 ?100%	 ?of	 ?the	 ?offspring	 ?as	 ?well	 ?as	 ?cleft	 ?soft	 ?palate	 ?(Xu	 ?et	 ?al.,	 ?2006).	 ?Here	 ?the	 ?mechanism	 ?underlying	 ?the	 ?submucous	 ?cleft	 ?phenotype	 ?included	 ?excessive	 ?cell	 ?proliferation	 ?and	 ?lack	 ?of	 ?apoptosis.	 ?This	 ?allowed	 ?the	 ?midline	 ?epithelial	 ?seam	 ?to	 ?persist	 ?instead	 ?of	 ?degrade.	 ?Interestingly,	 ?the	 ? 	 ? 57	 ?expression	 ?of	 ?the	 ?transcription	 ?factor	 ?Irf6	 ?was	 ?lacking	 ?in	 ?the	 ?epithelium.	 ?Mutations	 ?in	 ?the	 ?gene	 ?IRF6	 ?cause	 ?Van	 ?der	 ?Woude?s	 ?Syndrome	 ?(Kondo	 ?et	 ?al.,	 ?2002).	 ?	 ?Thus	 ?decreased	 ?IRF6	 ?levels	 ?could	 ?be	 ?contributing	 ?to	 ?non-??syndromic	 ?submucous	 ?clefts	 ?or	 ?other	 ?microforms	 ?of	 ?cleft	 ?palate.	 ?Recently	 ?TGFBR1	 ?and	 ?TGBR2	 ?mutations	 ?have	 ?been	 ?shown	 ?to	 ?cause	 ?Loeys-??Dietz	 ?syndrome	 ?(Breckpot	 ?et	 ?al.,	 ?2010;	 ?Cardoso	 ?et	 ?al.,	 ?2012;	 ?Loeys	 ?et	 ?al.,	 ?2005;	 ?Loeys	 ?and	 ?Dietz,	 ?1993;	 ?Singh	 ?et	 ?al.,	 ?2006).	 ?A	 ?striking	 ?feature	 ?of	 ?the	 ?syndrome	 ?is	 ?bivid	 ?uvula	 ?and/or	 ?cleft	 ?palate.	 ?This	 ?is	 ?all	 ?the	 ?more	 ?reason	 ?to	 ?investigate	 ?the	 ?expression	 ?of	 ?the	 ?receptors	 ?for	 ?TGF?	 ?in	 ?the	 ?soft	 ?palate.	 ?Downstream	 ?of	 ?TGF?	 ?signaling	 ?is	 ?the	 ?transcription	 ?factor	 ?Smad4.	 ?In	 ?conditional	 ?mouse	 ?knockouts	 ?of	 ?Smad4	 ?in	 ?the	 ?ectoderm	 ?there	 ?is	 ?no	 ?palate	 ?phenotype.	 ?However	 ?when	 ?these	 ?mice	 ?are	 ?crossed	 ?to	 ?the	 ?mice	 ?heterozygous	 ?for	 ?a	 ?loss-??of-??function	 ?mutation	 ?in	 ?Irf6,	 ?100%	 ?of	 ?the	 ?embryos	 ?have	 ?submucous	 ?clefts	 ?(Iwata	 ?et	 ?al.,	 ?2013).	 ?This	 ?is	 ?possibly	 ?the	 ?most	 ?penetrant	 ?cleft	 ?affecting	 ?the	 ?soft	 ?palate	 ?that	 ?has	 ?been	 ?reported	 ?in	 ?a	 ?mouse	 ?model.	 ?	 ?In	 ?the	 ?Tbx22	 ?knockout	 ?mouse	 ?mutant	 ?(Tbx22-??/-??)(Pauws	 ?et	 ?al.,	 ?2009a)	 ?fetuses	 ?exhibited	 ?a	 ?translucent	 ?area	 ?in	 ?the	 ?posterior	 ?palate.	 ?There	 ?was	 ?severely	 ?reduced	 ?bone	 ?formation	 ?in	 ?the	 ?posterior	 ?palate,	 ?despite	 ?the	 ?fact	 ?that	 ?the	 ?palatal	 ?shelves	 ?had	 ?fully	 ?fused.	 ?	 ?Some	 ?mutants	 ?even	 ?exhibited	 ?complete	 ?cleft	 ?palate,	 ?whereas	 ?others	 ?displayed	 ?submucous	 ?cleft	 ?palate	 ?with	 ?reduced	 ?bone	 ?formation	 ?and	 ?delayed	 ?osteoblast	 ?differentiation.	 ?	 ?	 ?As	 ?mentioned	 ?previously	 ?human	 ?mutations	 ?in	 ?TBX22	 ?cause	 ?X-??linked	 ?ankyloglossia	 ?and	 ?cleft	 ?palate	 ?which	 ?further	 ?supports	 ?a	 ?role	 ?for	 ?TBX22	 ?in	 ?soft	 ?palate	 ?morphogenesis.	 ? 	 ? 58	 ?Another	 ?member	 ?of	 ?the	 ?T-??box	 ?family,	 ?Tbx1,	 ?is	 ?also	 ?considered	 ?to	 ?be	 ?a	 ?major	 ?candidate	 ?gene	 ?for	 ?the	 ?pathogenesis	 ?of	 ?22q11.2	 ?deletion	 ?syndrome	 ?in	 ?humans	 ?(also	 ?known	 ?as	 ?DiGeorge	 ?syndrome	 ?or	 ?velo-??cardio-??facial	 ?syndrome).	 ?	 ?DiGeorge	 ?or	 ?22q11.2	 ?deletion	 ?syndrome	 ?(OMIM	 ?#188400)	 ?has	 ?an	 ?incidence	 ?of	 ?approximately	 ?1/4000	 ?live	 ?human	 ?births	 ?and	 ?commonly	 ?the	 ?patients	 ?will	 ?exhibit	 ?complete	 ?cleft	 ?palate	 ?or	 ?submucous	 ?cleft	 ?and	 ?velopharyngeal	 ?insufficiency	 ?(Scambler,	 ?2000).	 ?	 ?In	 ?the	 ?Tbx1-??/-??	 ?mouse	 ?mutant	 ?(Funato	 ?et	 ?al.,	 ?2012),	 ?all	 ?of	 ?the	 ?null	 ?mutants	 ?displayed	 ?an	 ?array	 ?of	 ?palatal	 ?deformities	 ?including	 ?compete	 ?clefts	 ?(41%),	 ?anterior	 ?clefts	 ?and	 ?an	 ?incomplete	 ?cleft	 ?palate	 ?(47%),	 ?and	 ?anterior	 ?clefts	 ?and	 ?soft	 ?palate	 ?clefts	 ?(12%).	 ?None	 ?of	 ?the	 ?wild	 ?type	 ?or	 ?Tbx1+/-??	 ?haploinsufficient	 ?mutants	 ?had	 ?any	 ?form	 ?of	 ?clefting.	 ?	 ?Whole	 ?mount	 ?staining	 ?showed	 ?high	 ?expression	 ?of	 ?Tbx1	 ?in	 ?the	 ?medial	 ?edge	 ?epithelium	 ?of	 ?the	 ?palatal	 ?shelves	 ?prior	 ?to	 ?fusion	 ?suggesting	 ?that	 ?this	 ?transcription	 ?factor	 ?is	 ?mediating	 ?signaling	 ?during	 ?epithelial	 ?adhesion	 ?and	 ?fusion.	 ?	 ?	 ?4.5  Limitations of this study 	 ?Due	 ?to	 ?the	 ?length	 ?of	 ?time	 ?the	 ?human	 ?specimens	 ?in	 ?my	 ?study	 ?spent	 ?in	 ?storage	 ?media,	 ?it	 ?was	 ?difficult	 ?to	 ?get	 ?any	 ?useable	 ?results	 ?from	 ?molecular	 ?tests.	 ?	 ?Pilot	 ?tests	 ?were	 ?performed	 ?using	 ?TUNEL	 ?assays	 ?and	 ?PCNA	 ?(Proliferating	 ?Nuclear	 ?Cell	 ?Antigen),	 ?however	 ?no	 ?information	 ?was	 ?obtained.	 ?Future	 ?directions	 ?for	 ?this	 ?project	 ?will	 ?involve	 ?performing	 ?TUNEL	 ?and	 ?PCNA	 ?on	 ?recently	 ?collected	 ?specimens	 ?that	 ?were	 ?tested	 ?for	 ?RNA	 ?viability	 ?and	 ?stored	 ?in	 ?70%	 ?EtOH.	 ?TUNEL	 ?will	 ?show	 ?if	 ?apoptosis	 ?is	 ?taking	 ?place	 ? 	 ? 59	 ?in	 ?the	 ?midline	 ?of	 ?the	 ?soft	 ?palate	 ?to	 ?degrade	 ?the	 ?midline	 ?epithelial	 ?seam.	 ?PCNA	 ?or	 ?another	 ?cell	 ?cycle	 ?antibody	 ?would	 ?indicate	 ?the	 ?pattern	 ?of	 ?cell	 ?proliferation	 ?in	 ?the	 ?developing	 ?soft	 ?palate.	 ?	 ?	 ? Based	 ?on	 ?the	 ?work	 ?of	 ?the	 ?previously	 ?discussed	 ?mouse	 ?models	 ?of	 ?submucous	 ?clefting,	 ?there	 ?are	 ?a	 ?number	 ?of	 ?candidate	 ?genes	 ?that	 ?would	 ?be	 ?valuable	 ?to	 ?create	 ?probes	 ?for	 ?to	 ?use	 ?in	 ?radioactive	 ?in-??situ	 ?hybridizations.	 ?	 ?These	 ?include	 ?BMPR1A,	 ?TGFBR2,	 ?TBX22,	 ?TGFB3,	 ?IRF6	 ?and	 ?TBX1.	 ?	 ? Another	 ?limitation	 ?of	 ?my	 ?study	 ?was	 ?that	 ?it	 ?was	 ?sometimes	 ?difficult	 ?to	 ?differentiate	 ?the	 ?epithelium	 ?from	 ?the	 ?mesenchyme	 ?in	 ?the	 ?soft	 ?palate.	 ?Thus	 ?I	 ?was	 ?not	 ?able	 ?to	 ?reliably	 ?score	 ?the	 ?presence	 ?of	 ?epithelial	 ?islands	 ?in	 ?all	 ?of	 ?the	 ?sections.	 ?In	 ?future	 ?work	 ?it	 ?would	 ?be	 ?advantageous	 ?to	 ?use	 ?a	 ?pan-??cytokeratin	 ?antibody	 ?which	 ?works	 ?well	 ?to	 ?detect	 ?epithelium	 ?in	 ?a	 ?wide	 ?variety	 ?of	 ?species.	 ?I	 ?believe	 ?this	 ?antibody	 ?would	 ?be	 ?robust	 ?enough	 ?to	 ?work	 ?on	 ?previously	 ?collected	 ?human	 ?samples.	 ?	 ?	 ?The	 ?Winsurf	 ?3D	 ?reconstruction	 ?program	 ?could	 ?have	 ?been	 ?used	 ?to	 ?measure	 ?area	 ?of	 ?each	 ?segmented	 ?region	 ?and	 ?then	 ?when	 ?combined	 ?with	 ?spacing	 ?between	 ?sections	 ?it	 ?would	 ?have	 ?been	 ?possible	 ?to	 ?calculate	 ?volume	 ?changes	 ?during	 ?development.	 ?These	 ?quantitative	 ?data	 ?would	 ?have	 ?provided	 ?additional	 ?insights	 ?into	 ?the	 ?dramatic	 ?growth	 ?of	 ?the	 ?soft	 ?palate.	 ?	 ?Finally	 ?I	 ?would	 ?like	 ?to	 ?increase	 ?the	 ?number	 ?of	 ?samples	 ?in	 ?my	 ?study	 ?and	 ?to	 ?investigate	 ?human	 ?fetuses	 ?from	 ?other	 ?populations.	 ?This	 ?would	 ?add	 ?more	 ?weight	 ?to	 ?my	 ?study	 ?since	 ?it	 ?would	 ?show	 ?that	 ?fusion	 ?is	 ?a	 ?widespread	 ?phenomenon	 ?that	 ?is	 ?not	 ?restricted	 ?to	 ?European	 ?ethnicity.	 ?It	 ?would	 ?also	 ?allow	 ?me	 ?to	 ?utilize	 ?a	 ?statistical	 ?power	 ? 	 ? 60	 ?analysis	 ?to	 ?determine	 ?mathematically	 ?if	 ?my	 ?hypothesis	 ?is	 ?true.	 ?It	 ?would	 ?involve	 ?grouping	 ?specimens	 ?of	 ?the	 ?same	 ?or	 ?similar	 ?ages	 ?to	 ?create	 ?multiple	 ?groups	 ?which	 ?could	 ?be	 ?used	 ?to	 ?compare	 ?seam	 ?development	 ?at	 ?different	 ?timepoints,	 ?and	 ?gain	 ?more	 ?definitive	 ?results	 ?of	 ?what	 ?a	 ?soft	 ?palate	 ?seam	 ?should	 ?look	 ?like	 ?at	 ?a	 ?given	 ?age	 ?(eg	 ?54-??57	 ?days,	 ?59-??64	 ?days,	 ?67-??70	 ?days	 ?and	 ?74+days).	 ?One	 ?possible	 ?test	 ?to	 ?use	 ?would	 ?be	 ?the	 ?Chi-??squared	 ?distribution	 ?to	 ?compare	 ?groups	 ?and	 ?validate	 ?my	 ?hypothesis.	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ?	 ? 	 ? 61	 ?References 	 ?Abbott, B. 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